Il-1 binding proteins

ABSTRACT

The disclosure provides binding proteins that specifically bind to IL-1α and IL-1β. These binding proteins can be organized into DVD-Igs. These proteins can be used to modulate the activity of IL-1α and/or IL-1β and can be used for the treatment immunological diseases such as rheumatoid arthritis, osteoarthritis, psoriasis, multiple sclerosis, and other autoimmune diseases. In addition, their uses in the amelioration and/or treatment of pain in an individual suffering from a disease or disorder associated with IL-1 accumulation.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent Application Ser. No. 61/670,834, filed Jul. 12, 2012, which is incorporated herein by reference in its entirety.

FIELD

The present disclosure provides IL-1 binding proteins, and specifically their uses in the prevention and/or treatment of acute and chronic immunological diseases such as rheumatoid arthritis, osteoarthritis, psoriasis, multiple sclerosis, and other autoimmune diseases. Uses of the IL-1 binding proteins in the amelioration and/or treatment of pain in an individual suffering from a disease or disorder associated with IL-1 accumulation are also described.

BACKGROUND

Cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF), are molecules produced by a variety of cells, such as monocytes and macrophages, that are mediators of inflammatory processes. Interleukin-1 is a cytokine with a wide range of biological and physiological effects, including fever, prostaglandin synthesis (e.g., fibroblasts, muscle cells and endothelial cells), T-lymphocyte activation, and interleukin-2 production.

Both IL-1α and IL-1β are produced by macrophages, monocytes and dendritic cells. They form an important part of the inflammatory response of the body against infection. These cytokines increase the expression of adhesion factors on endothelial cells to enable transmigration of leukocytes, the cells that fight pathogens, to sites of infection and re-set the hypothalamus thermoregulatory center, leading to an increased body temperature which expresses itself as fever. IL-1 is therefore called an endogenous pyrogen. The increased body temperature helps the body's immune system to fight infection. IL-1 is also important in the regulation of hematopoiesis. IL-1β production in peripheral tissue has also been associated with hyperalgesia (increased sensitivity to pain) associated with fever (Morgan et al., Brain Res., 1022 (1-2): 96-100 (2004)). For the most part, these two forms of IL-1 bind to the same cellular receptor. This receptor is composed of two related, but non-identical, subunits that transmit intracellular signals via a pathway that is mostly shared with certain other receptors. These include the Toll family of innate immune receptors and the receptor for IL-18. IL-1α and IL-1β also possess similar biological properties, including induction of fever, slow wave sleep, and neutrophilia, T- and B-lymphocyte activation, fibroblast proliferation, cytotoxicity for certain cells, induction of collagenases, synthesis of hepatic acute phase proteins, and increased production of colony stimulating factors and collagen.

cDNAs encoding the two distinct forms of IL-1 have been isolated and expressed; these cDNAs represent two different gene products, termed IL-1β (Auron et al., Proc. Natl. Acad. Sci. USA, 81: 7907-7911 (1984)) and IL-1α (Lomedico et al., Nature, 312: 458-462 (1984)). IL-1β is the predominant form produced by human monocytes both at the mRNA and protein levels. The two forms of human IL-1 share only 26% amino acid homology. Despite their distinct polypeptide sequences, the two forms of IL-1 have structural similarities (Auron et al., J. Mol. Cell. Immunol., 2: 169-177 (1985)), in that the amino acid homology is confined to discrete regions of the IL-1 molecule.

IL-1α and IL-1β are produced as precursor peptides. In other words they are made as a long protein that is then processed to release a shorter, active molecule, which is called the mature protein. Mature IL-1β, for example, is released from Pro-IL-1β following cleavage by a certain member of the caspase family of proteins, called caspase-1 or the interleukin-1 converting enzyme (ICE). The 3-dimensional structure of the mature forms of each member of the human IL-1 superfamily is composed of 12-14 β-strands producing a barrel-shaped protein.

Although a variety of antibodies to IL-1 have been described in the nearly two decades of work since the discovery of this critical proinflammatory cytokine, there remains a need for improved binding proteins that can effectively mediate or neutralize the activity of IL-1 in the inflammatory response and autoimmune disorders and for use in detecting IL-1β in samples and tissues.

SUMMARY

This disclosure provides proteins that bind human IL-1α and IL-1β. Binding proteins of the disclosure include but are not limited to antibodies, antigen binding portions thereof, and multivalent, multispecific binding proteins such as DVD-Ig™ binding proteins that can bind human IL-1α and IL-1β. The disclosure also provides methods of making and using the IL-1α and IL-1β binding proteins described herein as well as various compositions that may be used in methods of detecting IL-1α and IL-1β in a sample or in methods of treating or preventing a disorder in an individual that is associated with or suspected to be associated with IL-1 activity.

The disclosure also provides a binding protein comprising an antigen binding domain, wherein the binding protein is capable of binding human IL-1β and the antigen-binding domain comprises six CDRs, i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, as defined below: CDR-H1: X₁-X₂-G-V-S (SEQ ID NO:1), wherein; X₁ is D or E and X₂ is Y or F; CDR-H2: L-I-W-G-X₃-G-D-T-Y-Y-N (SEQ ID NO:2), wherein; X₃ is S or G; CDR-H3: Q-X₄-N-X₅-W-X₆-Y-D-L-Y-X₇-M-D-Y (SEQ ID NO:3), wherein; X₄ is T or R; X₅ is L or I; X₆ is A or G and X₇ is S or G; CDR-L1: Q-X₈-S-X₉-D-I-D-X₁₀-D-X₁₁-N (SEQ ID NO: 4), wherein; X₈ is A or T; X₉ is Q or T; X₁₀ is D or M; and X₁₁ is L or M; CDR-L2: X₁₂-X₁₃-X₁₄-X₁₅-L-X₁₆-P (SEQ ID NO: 5), wherein; X₁₂ is L or Q; X₁₃ is A or G; X₁₄ is S or M; X₁₅ is I, K or T; and X₁₆ is R, W or P; and CDR-L3: L-Q-X₁₇-D-X₁₈-X₁₉-P-L-T (SEQ ID NO: 6), wherein; X₁₇ is S or T; X₁₈ is S, W or R; and X₁₉ is F or L.

In an embodiment, an isolated binding protein described above comprises at least one CDR comprising an amino acid sequence selected from the group consisting of: residues 31-35 of SEQ ID NOs:30, 31, 32, 33 and 34; residues 50-65 of SEQ ID NOs:30, 31, 32, 33 and 34; residues 98-111 of SEQ ID NOs:30, 31, 32, 33 and 34; residues 24-34 of SEQ ID NOs: 36, 37, 38, 39 and 40; residues 50-56 of SEQ ID NOs: 36, 37, 38, 39 and 40; and residues 89-97 of SEQ ID NOs: 36, 37, 38, 39 and 40. In other embodiments, the binding protein described above can have 1, 2, 3, 4, 5, 6 or more CDRs. In various embodiments, these CDRs can comprise 1, 2, 3, 4, 5, 6 or more of the CDRs described above. In certain embodiments, the binding proteins comprise three CDRs from residues 31-35 of SEQ ID NOs:30, 31, 32, 33 and 34; residues 50-65 of SEQ ID NOs:30, 31, 32, 33 and 34; and residues 98-111 of SEQ ID NOs:30, 31, 32, 33 and 34; as well as three CDRs from residues 24-34 of SEQ ID NOs: 36, 37, 38, 39 and 40; residues 50-56 of SEQ ID NOs: 36, 37, 38, 39 and 40; and residues 89-97 of SEQ ID NOs: 36, 37, 38, 39 and 40. In other embodiments, the binding proteins comprise one CDR from each of residues 31-35 of SEQ ID NOs:30, 31, 32, 33 and 34; residues 50-65 of SEQ ID NOs:30, 31, 32, 33 and 34; residues 98-111 of SEQ ID NOs:30, 31, 32, 33 and 34; residues 24-34 of SEQ ID NOs: 36, 37, 38, 39 and 40; residues 50-56 of SEQ ID NOs: 36, 37, 38, 39 and 40; and residues 89-97 of SEQ ID NOs: 36, 37, 38, 39 and 40.

In another embodiment, the binding protein is capable of modulating a biological function of IL-1 or neutralizing IL-1. In another embodiment, the binding protein specifically binds both IL-1α and IL-1β. In another embodiment, the binding protein has an IC50 of between 30 and 60 pM using the MRC-5 bioassay.

In another embodiment, the binding protein according claim 1, further includes an IL-1α antigen binding domain comprising a CDR-H1 comprising an amino acid sequence of SEQ ID NO:132, a CDR-H2 comprising an amino acid sequence of SEQ ID NO:133, and a CDR-H3 comprising an amino acid sequence of SEQ ID NO:134, a CDR-L1 comprising an amino acid sequence of SEQ ID NO:135, a CDR-L2 comprising an amino acid sequence of SEQ ID NO:136, and a CDR-L3 comprising an amino acid sequence of SEQ ID NO:137.

In other embodiments, the binding protein is cleared from a CD-1 mouse subject at between 0.16 and 0.19 mL/h/kg when administered at 4-5 mg/kg, the volume of distribution of the binding protein is between 109 and 94 mL/kg when the binding protein is administered to a CD-1 mouse subject at 4-5 mg/kg and/or the half-life of the binding protein is between 19 and 14 days when the binding protein is administered to a CD-1 mouse subject at 4-5 mg/kg. In other embodiments, the binding protein is cleared from a Sprague-Dawley rat subject at 0.21 and 0.24 mL/h/kg when administered at 4-5 mg/kg; the volume of distribution of the binding protein is between 78 and 71 mL/kg when the binding protein is administered to a Sprague-Dawley rat subject at 4-5 mg/kg; and/or the half-life of the binding protein is between 19 and 12 days when the binding protein is administered to a Sprague-Dawley rat subject at 4-5 mg/kg.

The disclosure further provides a binding protein that specifically binds to both IL-1α and IL-1β, said binding protein comprising an IL-1β antigen binding domain comprising six CDRs:

CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, as defined below:

CDR-H1: X₁-X₂-G-V-S (SEQ ID NO:146), wherein;

-   -   X₁ is D, E, H, Y, N, G, V or Q and     -   X₂ is Y, F, D, N, H or S;

CDR-H2: X₃-I-X₄-X₅-X₆-G-X₇-T-X₈-Y-N-S-P-L-K-S (SEQ ID NO:147), wherein;

-   -   X₃ is L, V, R, G, F, W, M or I;     -   X₄ is W, G, L, Y, S, R, E or A;     -   X₅ is G, V, R, W, L, D or S;     -   X₆ is G, R, V, W, S, L, I, E or D;     -   X₇ is D, G, E, V, S, A, K, D, Y or Q; and     -   X₈ is Y, F, S, D, V, T or H;

CDR-H3: X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈-X₁₉-M-D-Y (SEQ ID NO:148), wherein;

-   -   X₉ is Q, D, K, P, H, A, Y, V or T;     -   X₁₀ is R, T, M, S, I, W, P, N or K;     -   X₁₁ is T, R, N, Y, W, V, M or K;     -   X₁₂ is L, M, R, I, G, W, Q, H F or A;     -   X₁₃ is W, S, G, R, V, L, K, A, Y or T;     -   X₁₄ is G, A, R, V, T, P, N, E or D;     -   X₁₅ is Y, F, N, D, H or S;     -   X₁₆ is D, Q, H, A, V, L or K;     -   X₁₇ is L, I, F, S, M or G;     -   X₁₈ is Y, N, D, V or D; and     -   X₁₉ is G, A, R, D or W;

CDR-L1: X₂₀-X₂₁-S-X₂₂-D-I-X₂₃-X₂₄-X₂₅-M-N (SEQ ID NO:149), wherein;

-   -   X₂₀ is I or Q;     -   X₂₁ is T or A;     -   X₂₂ is T or Q;     -   X₂₃ is D, P, Y, N, Q or L;     -   X₂₄ is V, P, A or F; and     -   X₂₅ is D, A, P, N, Y or V;

CDR-L2 have the sequence X₂₆-X₂₇-X₂₈-X₂₉-L-X₃₀-X₃₁ (SEQ ID NO:150), wherein;

-   -   X₂₆ is S or Y;     -   X₂₇ is Q, H, P, K or R;     -   X₂₈ is G or A;     -   X₂₉ is N or S;     -   X₃₀ is T, S, I, M or A; and     -   X₃₁ is P, L, S or T; and

CDR-L3: X₃₂-Q-X₃₃-X₃₄-X₃₅-X₃₆-X₃₇-L-T (SEQ ID NO:151), wherein;

-   -   X₃₂ is L or Q;     -   X₃₃ is 5, W, R, K, G or V;     -   X₃₄ is D, G, K or V;     -   X₃₅ is N, E, R, M or G;     -   X₃₆ is L, G, D or F; and     -   X₃₇ is P or A.

In another embodiment, the binding protein is capable of modulating a biological function of IL-1 or neutralizing IL-1. In another embodiment, the binding protein specifically binds both IL-1α and IL-1β. In another embodiment, the binding protein has an IC50 of between 30 and 60 pM using the MRC-5 bioassay.

In another embodiment, the binding protein further includes an IL-1α antigen binding domain comprising a CDR-H1 comprising an amino acid sequence of SEQ ID NO:132, a CDR-H2 comprising an amino acid sequence of SEQ ID NO:133, and a CDR-H3 comprising an amino acid sequence of SEQ ID NO:134, a CDR-L1 comprising an amino acid sequence of SEQ ID NO:135, a CDR-L2 comprising an amino acid sequence of SEQ ID NO:136, and a CDR-L3 comprising an amino acid sequence of SEQ ID NO:137.

In other embodiments, the binding protein is cleared from a CD-1 mouse subject at between 0.16 and 0.19 mL/h/kg when administered at 4-5 mg/kg, the volume of distribution of the binding protein is between 109 and 94 mL/kg when the binding protein is administered to a CD-1 mouse subject at 4-5 mg/kg and/or the half-life of the binding protein is between 19 and 14 days when the binding protein is administered to a CD-1 mouse subject at 4-5 mg/kg. In other embodiments, the binding protein is cleared from a Sprague-Dawley rat subject at 0.21 and 0.24 mL/h/kg when administered at 4-5 mg/kg; the volume of distribution of the binding protein is between 78 and 71 mL/kg when the binding protein is administered to a Sprague-Dawley rat subject at 4-5 mg/kg; and/or the half-life of the binding protein is between 19 and 12 days when the binding protein is administered to a Sprague-Dawley rat subject at 4-5 mg/kg.

The disclosure further provides a binding protein comprising first and second polypeptide chains, wherein said first polypeptide chain comprises a first VD1-(X1)n-VD2-C-(X2)_(n), wherein: VD1 is a first heavy chain variable domain; VD2 is a second heavy chain variable domain; C is a heavy chain constant domain; X1 is a linker with the proviso that it is not CH1; X2 is an Fc region; and n is independently 0 or 1; and wherein said second polypeptide chain comprises a second VD1-(X1)n-VD2-C-(X₂)n, wherein: VD1 is a first light chain variable domain; VD2 is a second light chain variable domain; C is a light chain constant domain; X1 is a linker with the proviso that it is not CH1; X2 does not comprise an Fc region; and n is independently 0 or 1; wherein, in said first polypeptide chain, VD1 comprises an amino acid sequence 95, 96, 97, 98, 99 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 31, 32, 33, 34, 420, 421, 422, 423, 424, 425, 426 and 427; and VD2 comprises an amino acid sequence 95, 96, 97, 98, 99 or 100% identical to the amino acid sequence SEQ ID NO: 128 wherein, in said second polypeptide chain, VD1 comprises an amino acid sequence 95, 96, 97, 98, 99 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 37, 38, 39, 40, 138, 139, 140, 141, 142, 143, 144 and 145; and VD2 comprises an amino acid sequence 95, 96, 97, 98, 99 or 100% identical to the amino acid sequence SEQ ID NO: 129.

In an embodiment, a binding protein described above comprises a first polypeptide chain that comprises an amino acid sequence 95, 96, 97, 98, 99 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 46, 48, 50, 52, and 54.

In another embodiment, a binding protein described above comprises a second polypeptide chain that comprises an amino acid sequence 95, 96, 97, 98, 99 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 47, 49, 51, 53, and 55.

In an embodiment, a binding protein of the disclosure described above comprises two first polypeptide chains and two second polypeptide chains.

In another aspect, in a binding protein described above X1 or X2 is an amino acid sequence selected from the group consisting of SEQ ID NOs: 42, 43, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 and 111. X1 or X2 can also be an amino acid sequence selected from the group consisting of SEQ ID NOs:42 and 43. In certain embodiments, X1 is an amino acid sequence comprising SEQ ID NO:42 and X2 is an amino acid sequence comprising SEQ ID NO:43.

In another embodiment, the disclosure provides a binding protein described above wherein the Fc region is selected from the group consisting of a native sequence Fc region and a variant sequence Fc region. In another embodiment, the Fc region is selected from the group consisting of an Fc region from an IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

The disclosure further provides a binding protein that specifically binds to both IL-1α and IL-1β, comprising first and second polypeptide chains, wherein said first polypeptide chain comprises a first VD1-(X1)n-VD2-C-(X2)n, wherein: VD1 is a first heavy chain variable domain; VD2 is a second heavy chain variable domain; C is a heavy chain constant domain; X1 is a linker with the proviso that it is not CH1; X2 is an Fc region; and n is independently 0 or 1; wherein said second polypeptide chain comprises a second VD1-(X1)n-VD2-C-(X2)n, wherein: VD1 is a first light chain variable domain; VD2 is a second light chain variable domain; C is a light chain constant domain; X1 is a linker with the proviso that it is not CH1; X2 does not comprise an Fc region; and n is independently 0 or 1; wherein, in said first polypeptide chain, VD1 comprises a VH amino acid sequence found in Table 3b; wherein, in said second polypeptide chain, comprises a VL amino acid sequence found in Table 3b; wherein the VD1 of the first polypeptide chain and the VD1 of the second polypeptide chain form a IL-1β antigen binding domain; and wherein the VD2 of the first polypeptide chain and the VD2 of the second polypeptide chain form a IL-1α antigen binding domain.

In another aspect, in a binding protein described above X1 or X2 is an amino acid sequence selected from the group consisting of SEQ ID NOs: 42, 43, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 and 111. X1 or X2 can also be an amino acid sequence selected from the group consisting of SEQ ID NOs:42 and 43. In certain embodiments, X1 is an amino acid sequence comprising SEQ ID NO:42 and X2 is an amino acid sequence comprising SEQ ID NO:43.

In another embodiment, the disclosure provides a binding protein described above wherein the Fc region is selected from the group consisting of a native sequence Fc region and a variant sequence Fc region. In another embodiment, the Fc region is selected from the group consisting of an Fc region from an IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

In another embodiment, the disclosure provides a binding protein conjugate that comprises a binding protein described above and further comprises an agent. Such agents include, but are not limited to, an immunoadhesion molecule, an imaging agent, a therapeutic agent, and a cytotoxic agent. Preferred imaging agents include, but are not limited to, a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. Preferred radiolabels include, but are not limited to, ³H ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho and ¹⁵³Sm. A preferred therapeutic or cytotoxic agent includes, but is not limited to, an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenic agent, an anti-mitotic agent, an anthracycline, toxin, and an apoptotic agent.

In one aspect, the disclosure provides a binding protein construct that comprises a binding protein described herein and further comprises a linker or an immunoglobulin constant domain. In an embodiment, the binding protein construct comprises a binding protein, wherein the binding protein is selected from the group consisting of: an immunoglobulin molecule, a disulfide linked Fv, a monoclonal antibody, an scFv, a chimeric antibody, a CDR-grafted antibody, a diabody, a humanized antibody, a multispecific antibody, an Fab, a dual specific antibody, a Fab′, a bispecific antibody, and a F(aN)₂, a DVD-Ig™, and an Fv.

In another embodiment, a binding protein conjugate comprises an agent that is a therapeutic or cytotoxic agent selected from the group consisting of: an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenic agent, an anti-mitotic agent, an anthracycline, toxin, and an apoptotic agent.

In an embodiment, a binding protein, binding protein construct, or binding protein conjugate described herein possesses a human glycosylation pattern.

Binding proteins, binding protein constructs, and binding protein conjugates described herein may exist as soluble proteins or as crystals. In an embodiment, such crystals are carrier-free pharmaceutical controlled released crystals. In another embodiment, a crystalline form of a binding protein, binding protein construct, or binding protein conjugate described herein has a greater in vivo half-life than its soluble counterpart. In another embodiment, a crystal of a binding protein, binding protein construct, or binding protein conjugate described herein retains biological activity of its soluble counterpart.

Compositions of the disclosure include a composition for the release of a crystallized binding protein, binding protein construct, or binding protein conjugate described herein, comprising:

(a) a formulation, wherein said formulation comprises a crystallized binding protein, binding protein construct, or binding protein conjugate described herein, and an ingredient; and

(b) at least one polymeric carrier.

Polymeric carriers useful in compositions of the disclosure include, without limitation, one or more of the group consisting of: poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutryate), poly (caprolactone), poly (dioxanone); poly (ethylene glycol), poly ((hydroxypropyl)methacrylamide, poly[(organo) phosphazene], poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends and copolymers thereof.

In another aspect, an ingredient of a composition of the disclosure is selected from the group consisting of albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol.

The disclosure also provides pharmaceutical compositions comprising a binding protein, a binding protein construct, or binding protein conjugate described herein, and a pharmaceutically acceptable carrier. Pharmaceutical compositions of the disclosure may further comprise at least one additional agent. In an embodiment, such an additional agent includes, but is not limited to, therapeutic agent, imaging agent, cytotoxic agent, angiogenesis inhibitors; kinase inhibitors; co-stimulation molecule blockers; adhesion molecule blockers; anti-cytokine antibody or functional fragment thereof; methotrexate; cyclosporin; rapamycin; FK506; detectable label or reporter; a TNF antagonist; an antirheumatic; a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteroid, an anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an immunosuppressive, a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an epinephrine or analog, a cytokine, and a cytokine antagonist.

In an embodiment, a pharmaceutical composition of the disclosure comprises a pharmaceutically acceptable carrier, wherein the carrier also serves as an adjuvant to increase the absorption or dispersion of the binding protein, binding protein construct, or binding protein conjugate in the composition. An exemplary adjuvant is hyaluronidase.

In an embodiment of the pharmaceutical, the binding protein conjugate comprises an imaging agent selected from the group consisting of: a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. In one aspect of this embodiment, said imaging agent is a radiolabel selected from the group consisting of: ³H ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho and ¹⁵³Sm.

In another embodiment, said binding protein conjugate comprises a therapeutic or cytotoxic agent selected from the group consisting of: an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenic agent, an anti-mitotic agent, an anthracycline, a toxin, and an apoptotic agent.

In an embodiment, the disclosure provides isolated nucleic acids encoding one or more amino acid sequences of a binding protein described herein. Such nucleic acids may be inserted into a vector for carrying out various genetic analyses or for expressing, characterizing, or improving one or more properties of a binding protein described herein. A vector may comprise a one or more nucleic acid molecules encoding one or more amino acid sequences of a binding protein described herein in which the one or more nucleic acid molecules is operably linked to appropriate transcriptional and/or translational sequences that permit expression of the binding protein in a particular host cell carrying the vector. Examples of vectors for cloning or expressing nucleic acids encoding amino acid sequences of binding proteins described herein include, but are not limited, pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, and pBJ.

The disclosure also provides a host cell comprising a vector comprising a nucleic acid encoding one or more amino acid sequences of a binding protein described herein. Host cells useful according to the disclosure may be prokaryotic or eukaryotic. An exemplary prokaryotic host cell is Escherichia coli. Eukaryotic cells useful as host cells according to the disclosure include protist cell, animal cell, plant cell, and fungal cell. An exemplary fungal cell is a yeast cell, including Saccharomyces cerevisiae. An exemplary animal cell useful as a host cell according to the disclosure includes, but is not limited to, a mammalian cell, an avian cell, and an insect cell. Preferred mammalian cells include CHO and COS cells. An insect cell useful as a host cell according to the disclosure is an insect Sf9 cell.

In another aspect, the disclosure provides a method of producing a binding protein described herein comprising culturing a host cell comprising a vector encoding the binding protein in culture medium under conditions sufficient to produce the binding protein capable of binding IL-1α and/or IL-1β. The protein so produced can be isolated and used in various compositions and methods described herein.

In another embodiment, the disclosure provides a method for reducing human IL-1 activity comprising contacting human IL-1 with a binding protein described herein, such that human IL-1 activity is reduced.

Another embodiment of the disclosure provides a method for treating a subject for a disorder by administering to the subject a binding protein described herein such that treatment is achieved.

In a further embodiment of a method of treatment described herein, the step of administering to the subject a binding protein or binding protein construct or binding protein conjugate described herein is by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intrao steal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intrapro static, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.

Another aspect of the disclosure is a method of treating a patient suffering from a disorder in which IL-1 is detrimental comprising the step of administering a binding protein, binding protein construct, or binding protein conjugate described herein before, concurrently with, or after the administration of a second agent, wherein the second agent is selected from the group consisting of inhaled steroids; beta-agonists; short-acting or long-acting beta-agonists; antagonists of leukotrienes or leukotriene receptors; ADVAIR; IgE inhibitors; anti-IgE antibodies; XOLAIR; phosphodiesterase inhibitors; PDE4 inhibitors; xanthines; anticholinergic drugs; mast cell-stabilizing agents; Cromolyn; IL-4 inhibitors; IL-5 inhibitors; eotaxin/CCR3 inhibitors; antagonists of histamine or its receptors including H1, H2, H3, and H4; antagonists of prostaglandin D or its receptors DP1 and CRTH2; TNF antagonists; a soluble fragment of a TNF receptor; ENBREL®; TNF enzyme antagonists; TNF converting enzyme (TACE) inhibitors; muscarinic receptor antagonists; TGF-beta antagonists; interferon gamma; perfenidone; chemotherapeutic agents, methotrexate; leflunomide; sirolimus (rapamycin) or an analog thereof, CCI-779; COX2 or cPLA2 inhibitors; NSAIDs; immunomodulators; p38 inhibitors; TPL-2, MK-2 and NFkB inhibitors; budenoside; epidermal growth factor; corticosteroids; cyclosporine; sulfasalazine; amino salicylates; 6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor antagonists; anti-IL-1β antibodies; anti-IL-6 antibodies; growth factors; elastase inhibitors; pyridinyl-imidazole compounds; antibodies or agonists of TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, EMAP-II, GM-CSF, FGF, or PDGF; antibodies of CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands; FK506; rapamycin; mycophenolate mofetil; ibuprofen; prednisolone; phosphodiesterase inhibitors; adenosine agonists; antithrombotic agents; complement inhibitors; adrenergic agents; IRAK, NIK, IKK, p38, or MAP kinase inhibitors; IL-1β converting enzyme inhibitors; TNF-aconverting enzyme inhibitors; T-cell signaling inhibitors; metalloproteinase inhibitors; 6-mercaptopurines; angiotensin converting enzyme inhibitors; soluble cytokine receptors; soluble p55 TNF receptor; soluble p75 TNF receptor; sIL-1RI; sIL-1RII; sIL-6R; anti-inflammatory cytokines; IL-4; IL-10; IL-11; and TGF-β.

Another aspect of the disclosure provides at least one IL-1 anti-idiotype antibody to at least one IL-1 binding protein described herein. The anti-idiotype antibody includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule such as, but not limited to, at least one CDR of a heavy or light chain or ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, that can be incorporated into a binding protein of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows line graphs depicting binding analyses of h1B12.1 scFv and rHC+LC M1S3-2 library output.

FIG. 2 shows VH sequences of h1B12.1 final variants aligned with h1B12 parental. From top to bottom the sequences are SEQ ID NOs:420, 31, 421, 422, 423, 424, 425, 33, 426, 34, 30, 32 and 427.

FIG. 3 shows VL sequences of the final affinity-matured variants (following sorting of h1B12.1 libraries) aligned with h1B12.1. From top to bottom the sequences are SEQ ID NOs:138, 37, 139, 140, 141, 142, 143, 39, 40, 144, 36, 38 and 145.

FIG. 4 is a line graph showing pharmacokinetic profiles of IL-1 α/β DVDs after a single 5 mg/kg IV dose in CD-1 mice.

FIG. 5 is a line graph showing pharmacokinetic profiles of IL-1 α/β DVDs after a single 4 mg/kg IV dose in SD rats.

DETAILED DESCRIPTION

This disclosure provides IL-1α and β binding proteins, including, but not limited to, anti-IL-1 α and/or β antibodies, or antigen-binding portions thereof, that bind IL-1α and/or β and multivalent, multispecific binding proteins such as DVD-Ig™ that bind IL-1β and/or IL-1α. Various aspects of the disclosure relate to antibodies and antibody fragments, DVD-Ig binding proteins, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such IL-1α/β binding proteins, including antibodies, DVD-Ig binding proteins, and fragments thereof. Methods of using the IL-1α/β binding proteins of the disclosure to detect human IL-1α/β; to inhibit human IL-1α/β, either in vitro or in vivo; and to regulate gene expression are also encompassed by the disclosure. The disclosure also encompasses any binding protein or antibody capable of competing with an IL-1α or β binding protein described herein.

In certain embodiments, this disclosure provides binding proteins that specifically bind IL-1α and/or IL-1β. In certain embodiments, the binding proteins have homology to VH and VL domains of antibodies that specifically bind IL-1α and/or IL-1β. In other embodiments, the binding proteins have sequences that correspond to the complementarity determining regions (CDRs) of these VH and VL domains. Each of the VH and VL domains contains three CDR domains: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. According to certain embodiments, the binding proteins described herein can contain 1, 2, 3, 4, 5 or 6 of these CDRs. One binding protein can comprise a sequence with high homology to a VH or a VL domain, or it can include sequences that are homologous to a VH and a VL domain in the same binding protein.

In certain embodiments, binding proteins with sequences homologous to CDR-H1 have the sequence X₁-X₂-G-V-S (SEQ ID NO:1), wherein; X₁ is D or E and X₂ is Y or F. In other embodiments, binding proteins with sequences homologous to CDR-H2 have the sequence L-I-W-G-X₃-G-D-T-Y-Y-N (SEQ ID NO:2), wherein; X₃ is S or G. In other embodiments, binding proteins with sequences homologous to CDR-H3 have the sequence Q-X₄-N-X₅-W-X₆-Y-D-L-Y-X₇-M-D-Y (SEQ ID NO:3), wherein; X₄ is T or R; X₅ is L or I; X₆ is A or G and X₇ is S or G. In other embodiments, binding proteins with sequences homologous to CDR-L1 have the sequence Q-X₈-S-X₉-D-I-D-X₁₀-D-X₁₁-N (SEQ ID NO:4), wherein; X₈ is A or T; X₉ is Q or T; X₁₀ is D or M; and X₁₁ is L or M. In other embodiments, binding proteins with sequences homologous to CDR-L2 have the sequence X₁₂-X₁₃-X₁₄-X₁₅-L-X₁₆-P (SEQ ID NO:5), wherein; X₁₂ is L or Q; X₁₃ is A or G; X₁₄ is S or M; X₁₅ is I, K or T; and X₁₆ is R, W or P. In other embodiments, binding proteins with sequences homologous to CDR-L3 have the sequence: L-Q-X₁₇-D-X₁₈-X₁₉-P-L-T (SEQ ID NO:6), wherein; X₁₇ is S or T; X₁₈ is 5, W or R; and X₁₉ is F or L.

According to certain embodiments, the binding proteins can include sequences homologous to the following CDR sequences in Table 1.

TABLE 1 Sequence of CDRs Domain Sequence Sequence Type Identifier 123456789012345678901234567890 CDR-H1 SEQ ID NO: 7 DYGVS CDR-H2 SEQ ID NO: 8 LIWGGGDTYYNSPLKS CDR-H3 SEQ ID NO: 9 QTNIWAYDLYSMDY CDR-H1 SEQ ID NO: 10 EFGVS CDR-H3 SEQ ID NO: 11 QRNLWAYDLYGMDY CDR-H2 SEQ ID NO: 12 LIWGSGDTYYNSPLKS CDR-H3 SEQ ID NO: 13 QTNLWAYDLYSMDY CDR-H3 SEQ ID NO: 14 QTNIWGYDLYGMDY CDR-H3 SEQ ID NO: 15 QTNLWAYDLYSMDY CDR-H1 SEQ ID NO: 132 MFGVH CDR-H2 SEQ ID NO: 133 AVSYDGSNKYYAESVKG CDR-H3 SEQ ID NO: 134 GRPKVVIPAPLAH CDR-L1 SEQ ID NO: 16 QTSTDIDDDLN CDR-L2 SEQ ID NO: 17 LASTLRP CDR-L3 SEQ ID NO: 18 LQSDRLPLT CDR-L1 SEQ ID NO: 19 QTSQDIDMDLN CDR-L2 SEQ ID NO: 20 QGSTLWP CDR-L3 SEQ ID NO: 21 LQTDSFPLT CDR-L1 SEQ ID NO: 22 QASQDIDDDLN CDR-L2 SEQ ID NO: 23 LASILRP CDR-L3 SEQ ID NO: 24 LQSDSFPLT CDR-L1 SEQ ID NO: 25 QASQDIDMDLN CDR-L2 SEQ ID NO: 26 QANTLPP CDR-L3 SEQ ID NO: 27 LQSDWLPLT CDR-L1 SEQ ID NO: 28 QASTDIDDDMN CDR-L2 SEQ ID NO: 29 LANKLRP CDR-L1 SEQ ID NO: 135 RASQGISSWLA CDR-L2 SEQ ID NO: 136 EASNLET CDR-L3 SEQ ID NO: 137 QQTSSFLLS

In other embodiments, binding proteins with sequences homologous to CDR-H1 have the sequence X₁-X₂-G-V-S (SEQ ID NO:146), wherein; X₁ is D, E, H, Y, N, G, V or Q and X₂ is Y, F, D, N, H or S. In other embodiments, binding proteins with sequences homologous to CDR-H2 have the sequence X₃-I-X₄-X₅-X₆-G-X₇-T-X₈-Y-N-S-P-L-K-S (SEQ ID NO:147), wherein; X₃ is L, V, R, G, F, W, M or I; X₄ is W, G, L, Y, S, R, E or A; X₅ is G, V, R, W, L, D or S; X₆ is G, R, V, W, S, L, I, E or D; X₇ is D, G, E, V, S, A, K, D, Y or Q; and X₈ is Y, F, S, D, V, T or H. In other embodiments, binding proteins with sequences homologous to CDR-H3 have the sequence X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈-X₁₉-M-D-Y (SEQ ID NO:148), wherein; X₉ is Q, D, K, P, H, A, Y, V or T; X₁₀ is R, T, M, S, I, W, P, N or K; X₁₁ is T, R, N, Y, W, V, M or K; X₁₂ is L, M, R, I, G, W, Q, H F or A; X₁₃ is W, S, G, R, V, L, K, A, Y or T; X₁₄ is G, A, R, V, T, P, N, E or D; X₁₅ is Y, F, N, D, H or S; X₁₆ is D, Q, H, A, V, L or K; X₁₇ is L, I, F, S, M or G; X₁₈ is Y, N, D, V or D; and X₁₉ is G, A, R, D or W. In other embodiments, binding proteins with sequences homologous to CDR-L1 have the sequence X₂₀-X₂₁-S-X₂₂-D-I-X₂₃-X₂₄-X₂₅-M-N (SEQ ID NO:149), wherein; X₂₀ is I or Q; X₂₁ is T or A; X₂₂ is T or Q; X₂₃ is D, P, Y, N, Q or L; X₂₄ is V, P, A or F; and X₂₅ is D, A, P, N, Y or V. In other embodiments, binding proteins with sequences homologous to CDR-L2 have the sequence X₂₆-X₂₇-X₂₈-X₂₉-L-X₃₀-X₃₁ (SEQ ID NO:150), wherein; X₂₆ is S or Y; X₂₇ is Q, H, P, K or R; X₂₈ is G or A; X₂₉ is N or S; X₃₀ is T, S, I, M or A; and X₃₁ is P, L, S or T. In other embodiments, binding proteins with sequences homologous to CDR-L3 have the sequence: X₃₂-Q-X₃₃-X₃₄-X₃₅-X₃₆-X₃₇-L-T (SEQ ID NO:151), wherein; X₃₂ is L or Q; X₃₃ is S, W, R, K, G or V; X₃₄ is D, G, K or V; X₃₅ is N, E, R, M or G; X₃₆ is L, G, D or F; and X37 is P or A.

In certain embodiments, the binding proteins disclosed herein contain sequences that have high homology to VH or VL domains of antibodies that specifically binds to IL-1α or IL-1β. These sequences can be 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to a VH or VL domain of an antibody that specifically binds to IL-1α or IL-1β. In other embodiments, the biding proteins described herein contain sequences that have high homology to VH or VL domains of antibodies that specifically binds to IL-1α or IL-1β wherein these sequences have the same number of amino acids as the VH or VL domain or fewer amino acids. Sequences that have fewer amino acids than the VH or VL domain—that they have identity to or have homology to—can have 121 or fewer amino acids. In other embodiments, the sequences have 107 or fewer amino acids. In other embodiments, the sequences have 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105 or fewer amino acids.

In certain embodiments, the binding proteins disclosed herein contain sequences that have high homology to VH or VL domains or fragments thereof, wherein the sequences are selected from those shown below in Table 2.

TABLE 2 Sequence of VH and VL Domains Domain Sequence Sequence Name Identifier 123456789012345678901234567890 13 VH SEQ ID NO: 30 EVQLQESGPGLVKPSETLSLTCTVSGFSLS DYGVSWIRQPPGKGLEWIGLIWGSGDTYYN SPLKSRLTISKDNSKSQVSLKLSSVTAADT AVYYCAKQTNIWAYDLYSMDYWGQGTLVTV SS 21 VH SEQ ID NO: 31 EVQLQESGPGLVKPSETLSLTCTVSGFSLS EFGVSWIRQPPGKGLEWIGLIWGGGDTYYN SPLKSRLTISKDNSKSQVSLKLSSVTAADT AVYYCAKQRNLWAYDLYGMDYWGQGTLVTV SS 34 VH SEQ ID NO: 32 EVQLQESGPGLVKPSETLSLTCTVSGFSLS DYGVSWIRQPPGKGLEWIGLIWGSGDTYYN SPLKSRLTISKDTSKSQVSLKLSSVTAADT AVYYCAKQTNLWAYDLYSMDYWGQGTLVTV SS A1 VH SEQ ID NO: 33 EVQLQESGPGLVKPSETLSLTCTVSGFSLR DYGVSWIRQPPGKGLEWLGLIWGSGDTYYN SPLKSRLTISKDTSKSQVSLKLSSVTAADT AVYYCAKQTNIWGYDLYGMDYWGQGTLVTV SS A3 VH SEQ ID NO: 34 EVQLQESGPGLVKPSETLSLTCTVSGFSLS DYGVSWIRQPPGKGLEWIGLIWGSGDTYYN SPLKSRLTISKDNSKSQVSLKLSSVTAADT AVYYCARQTNLWAYDLYSMDYWGQGTLVTV SS 1B12.9 SEQ ID NO: 35 EVQLQESGPGLVKPSETLSLTCTVSGFSLS VH DYGVSWIRQPPGKGLEWLGLIWGGGDTYYN SPLKSRLTISKDNSKSQVSLKLSSVTAADT AVYYCAKQRTLWGYDLYGMDYWGQGTLVTV SS X3 VH SEQ ID NO: 128 QVQLVESGGGVVQPGRSLRLSCTASGFTFS MFGVHWVRQAPGKGLEWVAAVSYDGSNKYY AESVKGRFTISRDNSKNILFLQMDSLRLED TAVYYCARGRPKVVIPAPLAHWGQGTLVTF SS 13 VL SEQ ID NO: 36 DIQMTQSPSSLSASVGDRVTITCQTSTDID DDLNWYQQKPGKAPKLLISLASTLRPGVPS RFSGSGSGTDFTFTISSLQPEDFATYYCLQ SDRLPLTFGQGTKLEIKR 21 VL SEQ ID NO: 37 DIQMTQSPSSLSASVGDRVTITCQTSQDID MDLNWYQQKPGKAPKLLISQGSTLWPGVPS RFSGSGSGTDFTFTISSLQPEDFATYYCLQ TDSFPLTFGQGTKLEIKR 34 VL SEQ ID NO: 38 DIQMTQSPSSLSASVGDRVTITCQASQDID DDLNWYQQKPGKAPKLLISLASILRPGVPS RFSGSGSGTDFTFTISSLQPEDFATYYCLQ SDSFPLTFGQGTKLEIKR A1 VL SEQ ID NO: 39 DIQMTQSPSSLSASVGDRVTITCQASQDID MDLNWYQQKPGKAPKLLISQANTLPPGVPS RFSGSGSGTDFTFTISSLQPEDFATYYCLQ SDWLPLTFGQGTKLEIKR A3 VL SEQ ID NO: 40 DIQMTQSPSSLSASVGDRVTITCQASTDID DDMNWYQQKPGKAPKLLISLANKLRPGVPS RFSGSGSGTDFTFTISSLQPEDFATYYCLQ TDSFPLTFGQGTKLEIKR 1B12.9 SEQ ID NO: 41 DTQMTQSPSSLSASVGDRVTITCITSTDID VL VDMNWYQQKPGKPPKLLISQGNTLRPGVPS RFSSSGSGTDFTFTISSLQPEDFATYYCLQ SDNLPLTFGQGTKLEIKR X3VL SEQID NO: 129 DIQMTQSPSSVSASVGDRVTITCRASQGIS SWLAWYQQKPGKAPKLLIYEASNLETGVPS RFSGSGSGSDFTLTISSLQPEDFATYYCQQ TSSFLLSFGGGTKVEHKR

In other embodiments, the VH or VL sequences can be selected from any of the mutants shown in Table 3a. Table 3a shows potential mutations at various positions of the h1B121 VH and VL sequences. Any one or more of the mutations can be made to construct a VH or VL domain that will specifically bind to IL-1β.

TABLE 3a H1B12 Heavy chain variable region 12345678901234567890123456789012345678901234567890123456789012 h1B12VH.1a EVQLQESGPGLVKPSETLSLTCTVSGFSLS DYGVS WIRQPPGKGLEWLG LIWGGGDTYYN (SEQ                           G  RED               I V GVR G F ID                              GHN                 R LRV E S NO: 428)                              NYH                 G YWW V D                              INS                 F SLS S V                              TGF                 W RDL A T                              MV                  M ESI K H                              KQ                  I A E Q                                                      D Y  SPLKS RLTISKDNSKSQVSLKLSSVTAADTAVYYCAK QRTLWGYDLYGMDY WGQGTLVTVSS        V T                          RTTKASAFQGNA                                      DMRMGRNKIDR                                      KSNRRVDHFVD                                      PIYIVTHASSW                                      HWWGLPSVM                                      APVWKN L                                      YNMQAE                                      VK HYD                                         FT H1B12 Light chain variable region 123456789012345678901234567890123456789012345678901234567890 h1B12VL.1a DTQVTQSPSSLSASVGDRVTITC ITSTDIDVDMN WYQQKPGKPPKLLI SQGNTLRP GVPS (SEQ I M QA Q PPAM A YHASS LL ID YAP P I PS NO: 429) NFN K M WT Q Y R A Q L V H H1B12 Heavy chain variable region 12345678901234567890123456789012345678901234567890123456789012 RFSSSGSGTDFTFTISSLQPEDFATYYC LQSDNLPLT FGQGTKLEIK    G                        Q WGEGA                               RKRD                               KVMF                               G G                               V

In other embodiments, the VH and/or VL sequences of a binding protein of the invention can be independently selected from Table 3b. Table 3b lists exemplary affinity-matured clones comprising mutations at one or more of the positions depicted in Table 3a supra.

TABLE 3b VH/VL sequences of affinity matured H1B12.1 clones VH SEQUENCE >Lib1_D4 (SEQ ID NO: 152) EVQLQESGPGLVKPSETLSLTCTVSGFSLGDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib2_H8 (SEQ ID NO: 153) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQTNLWGYDLYSMDYWGQ GTLVTVSS >Lib1_G2 (SEQ ID NO: 154) EVQLQESGPGLVKPSETLSLTCTVSGFSLSEFGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib1_B6 (SEQ ID NO: 155) EVQLQESGPGLVKPSETLSLTCTVSGFSLSNFGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_12E (SEQ ID NO: 156) EVQLQESGPGLVKPSETLSLTCTVSGFSLSGYGVSWIR QPPGKGLEWLGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib4_5C (SEQ ID NO: 157) EVQLQESGPGLVKPSETLSLTCTVSGFSLSKYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQTNTwAyDLySMDYWGQ GTLVTVSS >Lib2_H1 (SEQ ID NO: 158) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLwGyDmyGmDYWGQ GTLVTVSS >Lib4_12D(SEQ ID NO: 159) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDFGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQTNLWAYDLYSMDYWGQ GTLVTVSS >Lib1_H6 (SEQ ID NO: 160) EVQLQESGPGLVKPSETLSLTCTVSGFSLSGYGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_3A (SEQ ID NO: 161) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCARQRNIWAYDLYGMDYWGQ GTLVTVSS >Lib2_G5 (SEQ ID NO: 162) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQNNLWGYDLYGMDYWGQ GTLVTVSS >Lib2_B6 (SEQ ID NO: 163) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNVWGYDLYGMDYWGQ GTLVTVSS >Lib2_B1 (SEQ ID NO: 164) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKHRNIWGYDLYGMDYWGQ GTLVTVSS >Lib1_A7 (SEQ ID NO: 165) EVQLQESGPGLVKPSETLSLTCTVSGFSLSEFGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_6C (SEQ ID NO: 166) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDFGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib4_3 (SEQ ID NO: 167) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQSNVWGYDLYSMDYWGQ GTLVTVSS >Lib2_E9 (SEQ ID NO: 168) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQINIWGYDLYGMDYWGQ GTLVTVSS >Lib2_D6 (SEQ ID NO: 169) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCARQNTRWGDDLYGMDYWGQ GTLVTVSS >Lib4_29 (SEQ ID NO: 170) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYSMDYWGQ GTLVTVSS >Lib1_D8 (SEQ ID NO: 171) EVQLQESGPGLVKPSETLSLTCTVSGFSLSGYGVSWIR QPPGKGLEWLGLIWGSGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_72 (SEQ ID NO: 172) EVQLQESGPGLVKPSETLSLTCTVSGFSLRDYGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib4_11H (SEQ ID NO: 173) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQNNVWGYDLYGMDYWGQ GTLVTVSS >Lib2_H11 (SEQ ID NO: 174) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKHRNLWGYDLYGMDYWGQ GTLVTVSS >Lib4_58 (SEQ ID NO: 175) EVQLQESGPGLVKPSETLSLTCTVSGFSLSEYGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib4_5B (SEQ ID NO: 176) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCARQTNLWAYDLYSMDYWGQ GTLVTVSS >Lib2_H3 (SEQ ID NO: 177) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib1_D5 (SEQ ID NO: 178) EVQLQESGPGLVKPSETLSLTCTVSGFSLREYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib1_G7 (SEQ ID NO: 179) EVQLQESGPGLVKPSETLSLTCTVSGFSLIDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib4_2C (SEQ ID NO: 180) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQINLWGYDLYGMDYWGQ GTLVTVSS >Lib2_H10 (SEQ ID NO: 181) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib4_9C (SEQ ID NO: 182) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDFGVSWIR QPPGKGLEWLGLIWGSGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib1_A4 (SEQ ID NO: 183) EVQLQESGPGLVKPSETLSLTCTVSGFSLRDYGVSWIR QPPGKGLEWLGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib1_A3 (SEQ ID NO: 184) EVQLQESGPGLVKPSETLSLTCTVSGFSLRDYGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_31 (SEQ ID NO: 185) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQTNIWAYDLYSMDYWGQ GTLVTVSS >Lib2_H4 (SEQ ID NO: 186) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQTNVWGYDLYGMDYWGQ GTLVTVSS >Lib1_D1 (SEQ ID NO: 187) EVQLQESGPGLVKPSETLSLTCTVSGFSLSEFGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_86 (SEQ ID NO: 188) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDFGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQSNLwGyDLySmDYWGQ GTLVTVSS >Lib2_A6 (SEQ ID NO: 189) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNEwGyDLyGmDYWGQ GTLVTVSS >Lib4_10G (SEQ ID NO: 190) EVQLQESGPGLVKPSETLSLTCTVSGFSLKDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNIWGYDLYGMDYWGQ GTLVTVSS >Lib2_F1 (SEQ ID NO: 191) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCARQNNLWGYDLYGMDYWGQ GTLVTVSS >Lib4_7B (SEQ ID NO: 192) EVQLQESGPGLVKPSETLSLTCTVSGFSLSNYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNVWGYDLYGMDYWGQ GTLVTVSS >Lib2_D5 (SEQ ID NO: 193) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKNRNIWGYDLYGMDYWGQ GTLVTVSS >Lib4_84 (SEQ ID NO: 194) EVQLQESGPGLVKPSETLSLTCTVSGFSLMDYGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQTNLWAYDLYSMDYWGQ GTLVTVSS >Lib1_G11 (SEQ ID NO: 195) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_63 (SEQ ID NO: 196) EVQLQESGPGLVKPSETLSLTCTVSGFSLSEYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKNRNLWGYDLYSMDYWGQ GTLVTVSS >Lib1_F9 (SEQ ID NO: 197) EVQLQESGPGLVKPSETLSLTCTVSGFSLGEYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_6B (SEQ ID NO: 198) EVQLQESGPGLVKPSETLSLTCTVSGFSLSEFGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib4_10A (SEQ ID NO: 199) EVQLQESGPGLVKPSETLSLTCTVSGFSLRGYGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNIWGYDLYGMDYWGQ GTLVTVSS >Lib4_7 (SEQ ID NO: 200) EVQLQESGPGLVKPSETLSLTCTVSGFSLGDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQTNLWAYDLYSMDYWGQ GTLVTVSS >Lib4_1D (SEQ ID NO: 201) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCARQRNLWAYDLYTMDYWGQ GTLVTVSS >Lib4_61 (SEQ ID NO: 202) EVQLQESGPGLVKPSETLSLTCTVSGFSLRDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQTNLWAYDLYTMDYWGQ GTLVTVSS >Lib1_D11 (SEQ ID NO: 203) EVQLQESGPGLVKPSETLSLTCTVSGFSLTDYGVSWIR QPPGKGLEWIGMIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib1_E4 (SEQ ID NO: 204) EVQLQESGPGLVKPSETLSLTCTVSGFSLGEFGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_3B (SEQ ID NO: 205) EVQLQESGPGLVKPSETLSLTCTVSGFSLTEFGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQTNLWAYDLYSMDYWGQ GTLVTVSS >Lib4_10 (SEQ ID NO: 206) EVQLQESGPGLVKPSETLSLTCTVSGFSLSEYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQTNVWGYDLYGMDYWGQ GTLVTVSS >Lib1_D10 (SEQ ID NO: 207) EVQLQESGPGLVKPSETLSLTCTVSGFSLSEYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib2_H7 (SEQ ID NO: 208) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKNRNIWGVDLYGMDYWGQ GTLVTVSS >Lib1_D12 (SEQ ID NO: 209) EVQLQESGPGLVKPSETLSLTCTVSGFSLTDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib2_D9 (SEQ ID NO: 210) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCARQNNVWGYDLYGMDYWGQ GTLVTVSS >Lib4_1F (SEQ ID NO: 211) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib4_57 (SEQ ID NO: 212) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQNNVWGYDLYGMDYWGQ GTLVTVSS >Lib4_10H (SEQ ID NO: 213) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQTNLWGYDLYSMDYWGQ GTLVTVSS >Lib2_H5 (SEQ ID NO: 214) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQTNLWGYDLYGMDYWGQ GTLVTVSS >Lib4_1G (SEQ ID NO: 215) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib1_G6 (SEQ ID NO: 216) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib1_F8 (SEQ ID NO: 217) EVQLQESGPGLVKPSETLSLTCTVSGFSLKDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_69 (SEQ ID NO: 218) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQTNLWGYDLYGMDYWGQ GTLVTVSS >Lib4_4H (SEQ ID NO: 219) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQTNLWAYDLYSMDYWGQ GTLVTVSS >Lib4_12H (SEQ ID NO: 220) EVQLQESGPGLVKPSETLSLTCTVSGFSLTDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWAYDLYGMDYWGQ GTLVTVSS >Lib4_11 (SEQ ID NO: 221) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKVRNVWGYDLYTMDYWGQ GTLVTVSS >Lib1_C8 (SEQ ID NO: 222) EVQLQESGPGLVKPSETLSLTCTVSGFSLGDFGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_88 (SEQ ID NO: 223) EVQLQESGPGLVKPSETLSLTCTVSGFSLSEYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNIWGYDLYGMDYWGQ GTLVTVSS >Lib2_G10 (SEQ ID NO: 224) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQNNVWGYDLYGMDYWGQ GTLVTVSS >Lib4_37 (SEQ ID NO: 225) EVQLQESGPGLVKPSETLSLTCTVSGFSLSGYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib4_4A (SEQ ID NO: 226) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWAYDLYGMDYWGQ GTLVTVSS >Lib4_11G (SEQ ID NO: 227) EVQLQESGPGLVKPSETLSLTCTVSGFSLSEFGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQTNLwAyDLySMDYWGQ GTLVTVSS >Lib4_7C (SEQ ID NO: 228) EVQLQESGPGLVKPSETLSLTCTVSGFSLNDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRNIWGyDLyGmDYWGQ GTLVTVSS >Lib4_12F (SEQ ID NO: 229) EVQLQESGPGLVKPSETLSLTCTVSGFSLRDYGVSWIR QPPGKGLEWLGLIWGSGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib1_A10 (SEQ ID NO: 230) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib1_F3 (SEQ ID NO: 231) EVQLQESGPGLVKPSETLSLTCTVSGFSLSEYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_26 (SEQ ID NO: 232) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib2_G4 (SEQ ID NO: 233) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCARQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib4_65 (SEQ ID NO: 234) EVQLQESGPGLVKPSETLSLTCTVSGFSLMDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib2_F12 (SEQ ID NO: 235) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKNRNVWGYDMYAMDYWGQ GTLVTVSS >Lib4_66 (SEQ ID NO: 236) EVQLQESGPGLVKPSETLSLTCTVSGFSLRDFGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKVRNLWGYDMYTMDYWGQ GTLVTVSS >Lib1_E7 (SEQ ID NO: 237) EVQLQESGPGLVKPSETLSLTCTVSGFSLSEFGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib2_D7 (SEQ ID NO: 238) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQINLWGYDLYGMDYWGQ GTLVTVSS >Lib1_H4 (SEQ ID NO: 239) EVQLQESGPGLVKPSETLSLTCTVSGFSLSSYGVDWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_2A (SEQ ID NO: 240) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYSMDYWGQ GTLVTVSS >Lib4_11C (SEQ ID NO: 241) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWAYDLYGMDYWGQ GTLVTVSS >Lib4_12 (SEQ ID NO: 242) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDFGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQTNLWAYDLYSMDYWGQ GTLVTVSS >Lib1_G10 (SEQ ID NO: 243) EVQLQESGPGLVKPSETLSLTCTVSGFSLSHYGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_1A (SEQ ID NO: 244) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib4_2F (SEQ ID NO: 245) EVQLQESGPGLVKPSETLSLTCTVSGFSLREYGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKVRNLWGYDMYTMDYWGQ GTLVTVSS >Lib2_E2 (SEQ ID NO: 246) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWXQ GTLVTVSS >Lib1_F5 (SEQ ID NO: 247) EVQLQESGPGLVKPSETLSLTCTVSGFSLSEYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib1_F6 (SEQ ID NO: 248) EVQLQESGPGLVKPSETLSLTCTVSGFSLSNYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib1_C9 (SEQ ID NO: 249) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib1_F12 (SEQ ID NO: 250) EVQLQESGPGLVKPSETLSLTCTVSGFSLTDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib1_H12 (SEQ ID NO: 251) EVQLQESGPGLVKPSETLSLTCTVSGFSLGDYGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_96 (SEQ ID NO: 252) EVQLQESGPGLVKPSETLSLTCTVSGFSLSNYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCARQRNLWAYDLYTMDYWGQ GTLVTVSS >Lib2_F9 (SEQ ID NO: 253) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCARQRNIWGYDLYGMDYWGQ GTLVTVSS >Lib4_7E (SEQ ID NO: 254) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDFGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQTNIWGYDLYSMDYWGQ GTLVTVSS >Lib4_71 (SEQ ID NO: 255) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDFGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQTNLWAYDLYSMDYWGQ GTLVTVSS >Lib4_95 (SEQ ID NO: 256) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQSNIWAYDLYSMDYWGQ GTLVTVSS >Lib2_H9 (SEQ ID NO: 257) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCARQNNEWGYDLYGMDYWGQ GTLVTVSS >Lib2_A4 (SEQ ID NO: 258) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNIWGIDLYGMDYWGQ GTLVTVSS >Lib1_H11 (SEQ ID NO: 259) EVQLQESGPGLVKPSETLSLTCTVSGFSLDHYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib2_D1 (SEQ ID NO: 260) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNIWGVDMFGMDYWGQ GTLVTVSS >Lib4_50 (SEQ ID NO: 261) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQTNLWGYDLYGMDYWGQ GTLVTVSS >Lib1_G5 (SEQ ID NO: 262) EVQLQESGPGLVKPSETLSLTCTVSGFSLSKFGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_54 (SEQ ID NO: 263) EVQLQESGPGLVKPSETLSLTCTVSGFSLRDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQTNLWAYDLYSMDYWGQ GTLVTVSS >Lib1_B10 (SEQ ID NO: 264) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_8D (SEQ ID NO: 265) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCARQRNLWAYDLYTMDYWGQ GTLVTVSS >Lib1_C11 (SEQ ID NO: 266) EVQLQESGPGLVKPSETLSLTCTVSGFSLRDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_10F (SEQ ID NO: 267) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNIWGYDLYGMDYWGQ GTLVTVSS >Lib4_59 (SEQ ID NO: 268) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDFGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib1_C2 (SEQ ID NO: 269) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWEGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib1_H5 (SEQ ID NO: 270) EVQLQESGPGLVKPSETLSLTCTVSGFSLSECGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib2_F7 (SEQ ID NO: 271) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQTNVWGVDLYGMDYWGQ GTLVTVSS >Lib4_49 (SEQ ID NO: 272) EVQLQESGPGLVKPSETLSLTCTVSGFSLREYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQTNIWGYDLYSMDYWGQ GTLVTVSS >Lib4_8E (SEQ ID NO: 273) EVQLQESGPGLVKPSETLSLTCTVSGFSLSEFGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib1_E3 (SEQ ID NO: 274) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_6 (SEQ ID NO: 275) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib4_12G (SEQ ID NO: 276) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNIWGYDLYGMDYWGQ GTLVTVSS >Lib1_D2 (SEQ ID NO: 277) EVQLQESGPGLVKPSETLSLTCTVSGFSLGDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_9A (SEQ ID NO: 278) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDFGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWGFDLYGMDYWGQ GTLVTVSS >Lib2_D12 (SEQ ID NO: 279) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNIWGYDLYGMDYWXQ GTLVTVSS >Lib4_77 (SEQ ID NO: 280) EVQLQESGPGLVKPSETLSLTCTVSGFSLSEFGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWAYDLYGMDYWGQ GTLVTVSS >Lib4_51 (SEQ ID NO: 281) EVQLQESGPGLVKPSETLSLTCTVSGFSLKEYGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib4_9B (SEQ ID NO: 282) EVQLQESGPGLVKPSETLSLTCTVSGFSLTDYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYTMDYWGQ GTLVTVSS >Lib1_H9 (SEQ ID NO: 283) EVQLQESGPGLVKPSETLSLTCTVSGFSLSHYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib1_B2 (SEQ ID NO: 284) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDFGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS >Lib4_64 (SEQ ID NO: 285) EVQLQESGPGLVKPSETLSLTCTVSGFSLGEHGVSWIR QPPGKGLEWIGIIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib2_C7 (SEQ ID NO: 286) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QSPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWGYDLYGMDYWGQ GTLVTVSS >Lib4_6E (SEQ ID NO: 287) EVQLQESGPGLVKPSETLSLTCTVSGFSLGEYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDTSKS QVSLKLSSVTAADTAVYYCAKQRNIWGYDLYGMDYWGQ GTLVTVSS >Lib4_6F (SEQ ID NO: 288) EVQLQESGPGLVKPSETLSLTCTVSGFSLRDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQSNVWGYDLYSMDYWGQ GTLVTVSS >Lib4_4 (SEQ ID NO: 289) EVQLQESGPGLVKPSETLSLTCTVSGFSLSEYGVSWIR QPPGKGLEWIGLIWGSGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRNLWAYDLYGMDYWGQ GTLVTVSS >Lib2_B4 (SEQ ID NO: 290) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKHRNLWGFDLYGMDYWGQ GTLVTVSS >Lib2_G6 (SEQ ID NO: 291) EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIR QPPGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQTNIWGYDLYGMDYWGQ GTLVTVSS >Lib1_E6 (SEQ ID NO: 292) EVQLQESGPGLVKPSETLSLTCTVSGFSLSEYGVSWIR QPPGKGLEWIGLIWGGGDTYYNSPLKSRLTISKDNSKS QVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQ GTLVTVSS VL SEQUENCE >Lib4_8E (SEQ ID NO: 293) DIQMTQSPSSLSASVGDRVTITCQASQDIDEDLNWYQ QKPGKAPKLLISLASTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQTDSFPLTFGQGTKLEIK >Lib4_7 (SEQ ID NO: 294) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDMNWYQ QKPGKAPKLLISLANKLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDSLPLTFGQGTKLEIK >Lib3_C8 (SEQ ID NO: 295) DIQMTQSPSSLSASVGDRVTITCQTSQDIDDDMNWYQ QKPGKAPKLLISSASTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQKEKLPLTFGQGTKLEIK >Lib3_C2 (SEQ ID NO: 296) DIQMTQSPSSLSASVGDRVTITCQASTDIDFDLNWYQ QKPGKAPKLLISQASTLSSGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSEKLPLTFGQGTKLEIK >Lib3_A4 (SEQ ID NO: 297) DIQMTQSPSSLSASVGDRVTITCQTSTDIDEDLNWYQ QKPGKAPKLLISLANNLHPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQGDRLPLTFGQGTKLEIK >Lib3_D2 (SEQ ID NO: 298) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLGSTLQPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRLPLTFGQGTKLEIK >Lib4_7E (SEQ ID NO: 299) DIQMTQSPSSLSASVGDRVTITCQTSQDIDDDMNWYQ QKPGKAPKLLISLGSHLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRFPLTFGQGTKLEIK >Lib4_12 (SEQ ID NO: 300) DIQMTQSPSSLSASVGDRVTITCQTSQDIDEDLNWYQ QKPGKAPKLLISLASNLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRLPLTFGQGTKLEIK >Lib3_G9 (SEQ ID NO: 301) DIQMTQSPSSLSASVGDRVTITCQASQDIDVDLNWYQ QKPGKAPKLLISQANTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDWLPLTFGQGTKLEIK >Lib4_9B (SEQ ID NO: 302) DIQMTQSPSSLSASVGDRVTITCQASTDIDEDLNWYQ QKPGKAPKLLISLASTLPPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRFPLTFGQGTKLEIK >Lib3_C3 (SEQ ID NO: 303) DIQMTQSPSSLSASVGDRVTITCQTSQDIDEDLNWYQ QKPGKAPKLLISQASTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVERLPLTFGQGTKLEIK >Lib3_B3 (SEQ ID NO: 304) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDMNWYQ QKPGKAPKLLISLASSLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDGLPLTFGQGTKLEIK >Lib4_1D (SEQ ID NO: 305) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLASTLWPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDSLPLTFGQGTKLEIK >Lib3_E11 (SEQ ID NO: 306) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISQASTLGPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDRVPLTFGQGTKLEIK >Lib3_C6 (SEQ ID NO: 307) DIQMTQSPSSLSASVGDRVTITCQASQDIDEDMNWYQ QKPGKAPKLLISQANKLRSGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDRVPLTFGQGTKLEIK >Lib4_6C (SEQ ID NO: 308) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLGSTLQPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDSFPLTFGQGTKLEIK >Lib4_65 (SEQ ID NO: 309) DIQMTQSPSSLSASVGDRVTITCQTSTDIDDDMNWYQ QKPGKAPKLLISLASTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSSWLPLTFGQGTKLEIK >Lib4_10E (SEQ ID NO: 310) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDMNWYQ QKPGKAPKLLISLANKLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQTDSFPLTFGQGTKLEIK >Lib4_6E (SEQ ID NO: 311) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLASTLWPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDSFPLTFGQGTKLEIK >Lib3_E12 (SEQ ID NO: 312) DIQMTQSPSSLSASVGDRVTITCQTSTDIDDDLNWYQ QKPGKAPKLLISQGSTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDRVPLTFGQGTKLEIK >Lib4_66 (SEQ ID NO: 313) DIQMTQSPSSLSASVGDRVTITCQASQDIDMDMNWYQ QKPGKAPKLLISLGNILRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDSFPLTFGQGTKLEIK >Lib3_B9 (SEQ ID NO: 314) DIQMTQSPSSLSASVGDRVTITCQASQDIDVDMNWYQ QKPGKAPKLLISLASKLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVERFPLTFGQGTKLEIK >Lib4_72 (SEQ ID NO: 315) DIQMTQSPSSLSASVGDRVTITCQASTNIDDDMNWYQ QKPGKAPKLLISLANMLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQGDRLPLTFGQGTKLEIK >Lib3_E10 (SEQ ID NO: 316) DIQMTQSPSSLSASVGDRVTITCQASQDIDEDLNWYQ QKPGKAPKLLISLASTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDNLPLTFGQGTKLEIK >Lib4_9A (SEQ ID NO: 317) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDMNWYQ QKPGKAPKLLISLASTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDSLPLTFGQGTKLEIK >Lib4_20 (SEQ ID NO: 318) DIQMTQSPSSLSASVGDRVTITCQASQDIDEDLNWYQ QKPGKAPKLLISLANILRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRFPLTFGQGTKLEIK >Lib3_H10 (SEQ ID NO: 319) DIQMTQSPSSLSASVGDRVTITCITSTDIDDDMNWYQ QKPGKAPKLLISQASTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDRVPLTFGQGTKLEIK >Lib3_C7 (SEQ ID NO: 320) DIQMTQSPSSLSASVGDRVTITCQASQDIDMDLNWYQ QKPGKAPKLLISQGSTLWPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVERFPLTFGQGTKLEIK >Lib3_E9 (SEQ ID NO: 321) DIQMTQSPSSLSASVGDRVTITCQASTDIDMDLNWYQ QKPGKAPKLLISQANTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDRMPLTFGQGTKLEIK >Lib3_H5 (SEQ ID NO: 322) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDMNWYQ QKPGKAPKLLISQANMLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRLPLTFGQGTKLEIK >Lib4_64 (SEQ ID NO: 323) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDLNWYQ QKPGKAPKLLISLGSTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQTDSFPLTFGQGTKLEIK >Lib4_2B (SEQ ID NO: 324) DIQMTQSPSSLSASVGDRVTITCQASQDIDEDLNWYQ QKPGKAPKLLISLANILRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRLPLTFGQGTKLEIK >Lib4_10G (SEQ ID NO: 325) DIQMTQSPSSLSASVGDRVTITCQTSQDIDEDLNWYQ QKPGKAPKLLISLASTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDSFPLTFGQGTKLEIK >Lib3_D4 (SEQ ID NO: 326) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDLNWYQ QKPGKAPKLLISLGSTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRMPLTFGQGTKLEIK >Lib3_E2 (SEQ ID NO: 327) DIQMTQSPSSLSASVGDRVTITCQASTDIDVDMNWYQ QKPGKAPKLLISQGSTLPPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQIDRLPLTFGQGTKVEIKR >Lib4_8F (SEQ ID NO: 328) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDMNWYQ QKPGKAPKLLISLANKLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRLPLTFGQGTKLEIK >Lib3_G7 (SEQ ID NO: 329) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDLNWYQ QKPGKAPKLLISLGSSLWPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQGDSMPLTFGQGTKLEIK >Lib4_6 (SEQ ID NO: 330) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDMNWYQ QKPGKAPKLLISLANKLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRFPLTFGQGTKLEIK >Lib3_G11 (SEQ ID NO: 331) DIQMTQSPSSLSASVGDRVTITCQTSTDIDVDMNWYQ QKPGKAPKLLISLASTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRFPLTFGQGTKLEIK >Lib3_D7 (SEQ ID NO: 332) DIQMTQSPSSLSASVGDRVTITCQTSTDIDDDMNWYQ QKPGKAPKLLISQGSLLLPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCQQSDRLPLTFGQGTKLEIK >Lib3_A10 (SEQ ID NO: 333) DIQMTQSPSSLSASVGDRVTITCQASTDIDVDMNWYQ QKPGKAPKLLISLGNTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRLPLTFGQGTKLEIK >Lib4_6A (SEQ ID NO: 334) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDMNWYQ QKPGKAPKLLISLASTLWPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQGDSLPLTFGQGTKLEIK >Lib4_2 (SEQ ID NO: 335) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLANTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSSWLPLTFGQGTKLEIK >Lib3_D9 (SEQ ID NO: 336) DIQMTQSPSSLSASVGDRVTITCQTSTDIDDDLNWYQ QKPGKAPKLLISLASTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRVPLTFGQGTKLEIK >Lib4_54 (SEQ ID NO: 337) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLANTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDSLPLTFGQGTKLEIK >Lib3_C4 (SEQ ID NO: 338) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLANTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDKMPLTFGQGTKLEIK >Lib3_F7 (SEQ ID NO: 339) DIQMTQSPSSLSASVGDRVTITCQTSQDIDVDMNWYQ QKPGKAPKLLISLGSTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRLPLTFGQGTKLEIK >Lib3_F4 (SEQ ID NO: 340) DIQMTQSPSSLSASVGDRVTITCQTSTDIDFDMNWYQ QKPGKAPKLLISQGSMLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDRVPLTFGQGTKLEIK >Lib4_3 (SEQ ID NO: 341) DIQMTQSPSSLSASVGDRVTITXQASQDIDDDLNWYQ QKPGKAPKLLISLANTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRLPLTFGQGTKLEIK >Lib4_23 (SEQ ID NO: 342) DIQMTQSPSSLSASVGDRVTITCQXSQDIDDDLNWYQ QKPGKAPKLLISLASILRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDSFPLTFGQGTKLEIK >Lib3_H6 (SEQ ID NO: 343) DIQMTQSPSSLSASVGDRVTITCQASTDIDIDMNWYQ QKPGKAPKLLISLGSILRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCQQGDRFPLTFGQGTKLEIK >Lib4_73 (SEQ ID NO: 344) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLASILRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDSFPLTFGQGTKLEIK >Lib4_3D (SEQ ID NO: 345) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLASILRHGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDSFPLTFGQGTKLEIK >Lib3_G4 (SEQ ID NO: 346) DIQMTQSPSSLSASVGDRVTITCQASQDIDEDLNWYQ QKPGKAPKLLISLANILRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDMLPLTFGQGTKLEIK >Lib3_F10 (SEQ ID NO: 347) DIQMTQSPSSLSASVGDRVTITCQTSTDIDDDLNWYQ QKPGKAPKLLISQASTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDRVPLTFGQGTKLEIK >Lib4_5B (SEQ ID NO: 348) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDLNWYQ QKPGKAPKLLISLGSTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRLPLTFGQGTKLEIK >Lib3_B8 (SEQ ID NO: 349) DIQMTQSPSSLSASVGDRVTITCQTSTDIDVDMNWYQ QKPGKAPKLLISQASTLISGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVERLPLTFGQGTKLEIK >Lib4_6B (SEQ ID NO: 350) DIQMTQSPSSLSASVGDRVTITCQTSTDIDDDLNWYQ QKPGKAPKLLISLASKLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDSFPLTFGQGTKLEIK >Lib4_5C (SEQ ID NO: 351) DIQMTQSPSSLSASVGDRVTITCQASTDIDEDLNWYQ QKPGKAPKLLISLASTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQTDSFPLTFGQGTKLEIK >Lib4_12F (SEQ ID NO: 352) DIQMTQSPSSLSASVGDRVTITCQTSTDIDEDMNWYQ QKPGKAPKLLISLASMLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQGDRLPLTFGQGTKLEIK >Lib4_11 (SEQ ID NO: 353) DIQMTQSPSSLSASVGDRVTITCQASTDIDADLNWYQ QKPGKAPKLLISQANTLWPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDRFPLTFGQGTKLEIK >Lib3_D8 (SEQ ID NO: 354) DIQMTQSPSSLSASVGDRVTITCQASTDIDIDMNWYQ QKPGKAPKLLISLGSTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCQQGDKFPLTFGQGTKLEIK >Lib3_A8 (SEQ ID NO: 355) DIQMTQSPSSLSASVGDRVTITCQASTDIDMDLNWYQ QKPGKAPKLLISQASSLTPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVEKLPLTFGQGTKLEIK >Lib3_D11 (SEQ ID NO: 356) DIQMTQSPSSLSASVGDRVTITCQTSQDIDADLNWYQ QKPGKAPKLLISQASKLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVESLPLTFGQGTKLEIK >Lib4_1A (SEQ ID NO: 357) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLGSTLSPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDWLPLTFGQGTKLEIK >Lib3_B1 (SEQ ID NO: 358) DIQMTQSPSSLSASVGDRVTITCQTSTDIDDDLNWYQ QKPGKAPKLLISQASTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDRMPLTFGQGTKLEIK >Lib4_74 (SEQ ID NO: 359) DIQMTQSPSSLSASVGDRVTITCQVSQDIDDDLNWYQ QKPGKAPKLLISLASTLWPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQGDSLPLTFGQGTKLEIK >Lib3_F6 (SEQ ID NO: 360) DIQMTQSPSSLSASVGDRVTITCQTSTDIDVDMNWYQ QKPGKAPKLLISQANILSSGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVEKLPLTFGQGTKLEIK >Lib3_F8 (SEQ ID NO: 361) DIRMTQSPSSLSASVGDRVTITCQASQDIDMDLNWYQ QKPGKAPKLLISQASTLLPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQIDELPLTFGQGTKLEIK >Lib4_1F (SEQ ID NO: 362) DIQMTQSPSSLSASVGDRVTITCQASQDIDEDMNWYQ QKPGKAPKLLISQASTLGPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQTDSFPLTFGQGTKLEIK >Lib3_A5 (SEQ ID NO: 363) DIQMTQSPSSLSASVGDRVTITCITSQDIDEDLNWYQ QKPGKAPKLLISLASTLLPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRLPLTFGQGTKLEIK >Lib4_6H (SEQ ID NO: 364) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDLNWYQ QKPGKAPKLLISLASMLGPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQTDSFPLTFGQGTKLEIK >Lib4_10F (SEQ ID NO: 365) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDLNWYQ QKPGKAPKLLISLGSILRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCQQGDRFPLTFGQGTKLEIK >Lib3_H7 (SEQ ID NO: 366) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDLNWYQ QKPGKAPKLLISSANTLLPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQKEKLPLTFGQGTKLEIK >Lib4_88 (SEQ ID NO: 367) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLASTLWPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRFPLTFGQGTKLEIK >Lib4_77 (SEQ ID NO: 368) DIQMTQSPSSLSASVGDRVTITCQTSQDIDMDLNWYQ QKPGKAPKLLISQGSTLWPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQTDSFPLTFGQGTKLEIK >Lib4_4D (SEQ ID NO: 369) GIQMTQSPSSLSASVGDRVTITCQASQDIDDDMNWYQ QKPGKAPKLLISLASMLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDSFPLTFGQGTKLEIK >Lib4_12H (SEQ ID NO: 370) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLASILRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQIDSFPLTFGQGTKLEIK >Lib4_10H (SEQ ID NO: 371) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLASNLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDSFPLTFGQGTKLEIK >Lib3_E6 (SEQ ID NO: 372) DIQMTQSPSSLSASVGDRVTITCQASQDIDEDLNWYQ QKPGKAPKLLISQGSTLWSGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCQQGDRLPLTFGQGTKLEIK >Lib4_50 (SEQ ID NO: 373) DIQMTQSPSSLSASVGDRVTITCQASTDIDTDLNWYQ QKPGKAPKLLISQASTLFPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDWLPLTFGQGTKLEIK >Lib4_1B (SEQ ID NO: 374) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLASILRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDSFPLTFGQGTKLEIK >Lib4_76 (SEQ ID NO: 375) DIQMTQSPSSLSASVGDRVTITCQASTDIDVDLNWYQ QKPGKAPKLLISLGNILRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRLPLTFGQGTKLEIK >Lib4_7F (SEQ ID NO: 376) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLANTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRFPLTFGQGTKLEIK >Lib4_26 (SEQ ID NO: 377) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLASTLWPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDRFPLTFGQGTKLEIK >Lib4_69 (SEQ ID NO: 378) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLASNLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDWLPLTFGQGTKLEIK >Lib4_2H (SEQ ID NO: 379) DIQMTQSPSSLSASVGDRVTITCQTSTDIDDDLNWYQ QKPGKAPKLLISLASTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRLPLTFGQGTKLEIK >Lib4_49 (SEQ ID NO: 380) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDLNWYQ QKPGKAPKLLISLGSTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRFPLTFGQGTKLEIK >Lib3_F9 (SEQ ID NO: 381) DIQMTQSPSSLSASVGDRVTITCQTSTDIDVDLNWYQ QKPGKAPKLLISQASTLHTGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVERLPLTFGQGTKLEIK >Lib3_G8 (SEQ ID NO: 382) DIQMTQSPSSLSASVGDRVTITCQTSQDIDDDLNWYQ QKPGKAPKLLISQASTLFPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCQQGDRLPLTFGQGTKLEIK >Lib4_51 (SEQ ID NO: 383) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDLNWYQ QKPGKAPKLLISLANMLHPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRFPLTFGQGTKLEIK >Lib4_10A (SEQ ID NO: 384) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLASTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDSFPLTFGQGTKLEIK >Lib4_53 (SEQ ID NO: 385) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLGSTLQPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDWLPLTFGQGTKLEIK >Lib4_57 (SEQ ID NO: 386) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDLNWYQ QKPGKAPKLLISLANTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDSFPLTFGQGTKLEIK >Lib4_84 (SEQ ID NO: 387) DIQMTQSPSSLSASVGDRVTITCQTSQDIDDDMNWYQ QKPGKAPKLLISLASMLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRLPLTFGQGTKLEIK >Lib3_D3 (SEQ ID NO: 388) DIQMTQSPSSLSASVGDRVTITCQTSQDIDDDMNWYQ QKPGKAPKLLISQGSSLLPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDRVPLTFGQGTKLEIK >Lib4_3A (SEQ ID NO: 389) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLASTLWPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRLPLTFGQGTKLEIK >Lib3_C10 (SEQ ID NO: 390) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDMNWYQ QKPGKAPKLLISQASTLIPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDSFPLTFGQGTKLEIK >Lib4_29 (SEQ ID NO: 391) DIQMTQSPSSLSASVGDRVTITCQTSTDIDDDLNWYQ QKPGKAPKLLISLASTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDSFPLTFGQGTKLEIK >Lib4_37 (SEQ ID NO: 392) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLASTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRFPLTFGQGTKLEIK >Lib4_11C (SEQ ID NO: 393) DIQMTQSPSSLSASVGDRVTITCIASTDIDDDMNWYQ QKPGKAPKLLISLASILRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRLPLTFGQGTKLEIK >Lib4_95 (SEQ ID NO: 394) DIQMTQSPSSLSASVGDRVTITCQTSTDIDVDLNWYQ QKPGKAPKLLISLANTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRFPLTFGQGTKLEIK >Lib4_4 (SEQ ID NO: 395) DIQMTQSPSSLSASVGDRVTITCQTSQDIDDDMNWYQ QKPGKAPKLLISLGSTLWPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQGDSLPLTFGQGTKLEIK >Lib3_F11 (SEQ ID NO: 396) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLASTLWPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCQQGERFPLTFGQGTKLEIK >Lib4_8C (SEQ ID NO: 397) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLASTLWPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQGDSLPLTFGQGTKLEIK >Lib3_F3 (SEQ ID NO: 398) DIQMTQSPSSLSASVGDRVTITCQASTDIDEDMNWYQ QKPGKAPKLLISSANILPRGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQKERLPLTFGQGTKLEIK >Lib3_G5 (SEQ ID NO: 399) DIQMTQSPSSLSASVGDRVTITCQASQDIDMDMNWYQ QKPGKAPKLLISQASTLRSGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQTERFPLTFGQGTKLEIK >Lib4_55 (SEQ ID NO: 400) DIQMTQSPSSLSASVGDRVTITCQASTDIDADLNWYQ QKPGKAPKLLISLGSTLRPGVSSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDKFPLTFGQGTKLEIK >Lib3_D12 (SEQ ID NO: 401) DIQMTQSPSSLSASVGDRVTITCQASQDIDVDLNWYQ QKPGKAPKLLISLANTLHPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVERFPLTFGQGTKLEIK >Lib3_C11 (SEQ ID NO: 402) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDLNWYQ QKPGKAPKLLISHGSTLQHGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDRLPLTFGQGTKLEIK >Lib3_B7 (SEQ ID NO: 403) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDLNWYQ QKPGKAPKLLISLGNTLHPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCQQSDRLPLTFGQGTKLEIK >Lib4_46 (SEQ ID NO: 404) DIQMTQSPSSLSASVGDRVTITCQASQDIDEDLNWYQ QKPGKAPKLLISLASTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDSFPLTFGQGTKLEIK >Lib4_2C (SEQ ID NO: 405) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLANTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDSFPLTFGQGTKLEIK >Lib3_C9 (SEQ ID NO: 406) DIQMTQSPSSLSASVGDRVTITCQASTDIDEDLNWYQ QKPGKAPKLLISLANNLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQIERLPLTFGQGTKLEIK >Lib3_E8 (SEQ ID NO: 407) DIQMTQSPSSLSASVGDRVTITCQTSTDIDDDMNWYQ QKPGKAPKLLISLANNLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQCDRLPLTFGQGTKLEIK >Lib4_3B (SEQ ID NO: 408) DIQMTQSPSSLSASVGDRVTITCQVSQDIDDDLNWYQ QKPGKAPKLLISLGSTLQPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRLPLTFGQGTKLEIK >Lib4_10B (SEQ ID NO: 409) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDLNWYQ QKPGKAPKLLISLGSKLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDSFPLTFGQGTKLEIK >Lib4_86 (SEQ ID NO: 410) DIQMTQSPSSLSASVGDRVTITCQASTDIDEDLNWYQ QKPGKAPKLLISLASKLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRFPLTFGQGTKLEIK >Lib3_B10 (SEQ ID NO: 411) DIQMTQSPSSLSASVGDRVTITCQASTDIDVDLNWYQ QKPGKAPKLLISQASTLPPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDSLPLTFGQGTKLEIK >Lib4_63 (SEQ ID NO: 412) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDLNWYQ QKPGKAPKLLISLASTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRFPLTFGQGTKLEIK >Lib3_E7 (SEQ ID NO: 413) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDMNWYQ QKPGKAPKLLISLGSKLWPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDNLPLTFGQGTKLEIK >Lib3_D10 (SEQ ID NO: 414) DIQMTQSPSSLSASVGDRVTITCQASQDIDTDMNWYQ QKPGKAPKLLIYDANTLRSGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQKDRLPLTFGQGTKLEIK >Lib4_61 (SEQ ID NO: 415) DIQMTQSPSSLSASVGDRVTITCQASTDIDVDMNWYQ QKPGKAPKLLISLASKLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDSFPLTFGQGTKLEIK >Lib4_85 (SEQ ID NO: 416) DIQMTQSPSSLSASVGDRVTITCQASQDIDADLNWYQ QKPGKAPKLLISLASKLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQVDSFPLTFGQGTKLEIK >Lib4_1G (SEQ ID NO: 417) DIQMTQSPSSLSASVGDRVTITCQASQDIDDDMNWYQ QKPGKAPKLLISLASTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQGDSLPLTFGQGTKLEIK >Lib4_7D (SEQ ID NO: 418) DIQMTQSPSSLSASVGDRVTITCQASTDIDDDLNWYQ QKPGKAPKLLISLGSTLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDSLPLTFGQGTKLEIK >Lib4_28 (SEQ ID NO: 419) DIQMTQSPSSLSASVGDRVTITCQASTDIDEDLNWYQ QKPGKAPKLLISLANNLRPGVPSRFSGSGSGTDFTFT ISSLQPEDFATYYCLQSDRFPLTFGQGTKLEIK

In other embodiments, the binding proteins disclosed herein contain two or more sequences with homology to two or more VH or VL sequences or fragments thereof. The binding proteins disclosed herein can contain sequences with homology to one VH and one VL sequence or fragments thereof. In one embodiment, both of the VH and/or VL sequences specifically bind to the same target protein. In other embodiments, they bind to different epitopes on the same protein.

In another embodiment, the sequences homologous to two or more VH and/or VL sequences or fragments thereof in a binding protein described herein are joined by a linker. The linker can be an amino acid sequence between 5 and 100 amino acids in length. In other embodiments, the linker moiety is between 5 and 90, 5 and 80, 5 and 70, 5 and 60, 5 and 50, 5 and 40, 5 and 30, 5 and 20 and 5 and 10 amino acids in length. In certain specific embodiments, the linker sequences are selected from TVAAP (SEQ ID NO:42) and ASTKGP (SEQ ID NO:43). Additional examples of linkers are described below. Thus, in certain embodiments, formulas for the binding proteins described herein can include VH-Linker-VH, VH-Linker-VL and VL-Linker-VL, wherein VH and VL represent sequences with homology to VH and VL domains or fragments thereof.

In certain embodiments, the binding proteins described herein contain two or more sequences with homology to two or more VH sequences or fragments thereof. In certain embodiments, each of the VH sequences specifically binds to the same proteins. In this situation, the two VH sequences can bind to different epitopes on the same protein. In other embodiments, the binding proteins described herein contain two or more sequences with homology to two or more VL sequences or fragments thereof. In certain embodiments, each of the VL sequences specifically binds to the same proteins. In this situation, the two VL sequences can bind to different epitopes on the same protein. In certain embodiments, each of the VL sequences specifically binds to different proteins. The binding proteins described herein can also be larger protein structures including three or more VH and/or VL domains. For example, the binding protein may be a DVD-Ig. Examples of these binding proteins are shown in Table 4 and include so-called “X3” sequences which are VH and VL domains from an antibody that specifically binds IL-1α and inhibits IL-1α from binding to its receptor. The X3 antibody specifically binds to IL-1α but not to IL-1β. It is also able to inhibit native human IL-1α biological activity.

TABLE 4 Binding Proteins Sequence Sequence Name Identifier 123456789012345678901234567890 1B12.13. SEQ ID NO: EVQLQESGPGLVKPSETLSLTCTVSGFSLS SS.X3.VH 44 DYGVSWIRQPPGKGLEWIGLIWGSGDTYYN SPLKSRLTISKDNSKSQVSLKLSSVTAADT AVYYCAKQTNIWAYDLYSMDYWGQGTLVTV SSASTKGPQVQLVESGGGVVQPGRSLRLSC TASGFTFSMFGVHWVRQAPGKGLEWVAAVS YDGSNKYYAESVKGRFTISRDNSKNILFLQ MDSLRLEDTAVYYCARGRPKVVIPAPLAHW GQGTLVTFSS 1B12.13. SEQ ID NO: DIQMTQSPSSLSASVGDRVTITCQTSTDID SS.X3.VL 45 DDLNWYQQKPGKAPKLLISLASTLRPGVPS RFSGSGSGTDFTFTISSLQPEDFATYYCLQ SDRLPLTFGQGTKLEIKRTVAAPDIQMTQS PSSVSASVGDRVTITCRASQGISSWLAWYQ QKPGKAPKLLIYEASNLETGVPSRFSGSGS GSDFTLTISSLQPEDFATYYCQQTSSFLLS FGGGTKVEHKR 1B12.21. SEQ ID NO: EVQLQESGPGLVKPSETLSLTCTVSGFSLS SS.X3.VH 46 EFGVSWIRQPPGKGLEWIGLIWGGGDTYYN SPLKSRLTISKDNSKSQVSLKLSSVTAADT AVYYCAKQRNLWAYDLYGMDYWGQGTLVTV SSASTKGPQVQLVESGGGVVQPGRSLRLSC TASGFTFSMFGVHWVRQAPGKGLEWVAAVS YDGSNKYYAESVKGRFTISRDNSKNILFLQ MDSLRLEDTAVYYCARGRPKVVIPAPLAHW GQGTLVTFSS 1B12.21. SEQ ID NO: DIQMTQSPSSLSASVGDRVTITCQTSQDID SS.X3.VL 47 MDLNWYQQKPGKAPKLLISQGSTLWPGVPS RFSGSGSGTDFTFTISSLQPEDFATYYCLQ TDSFPLTFGQGTKLEIKRTVAAPDIQMTQS PSSVSASVGDRVTITCRASQGISSWLAWYQ QKPGKAPKLLIYEASNLETGVPSRFSGSGS GSDFTLTISSLQPEDFATYYCQQTSSFLLS FGGGTKVEHKR 1B12.34. SEQ ID NO: EVQLQESGPGLVKPSETLSLTCTVSGFSLS SS.X3.VH 48 DYGVSWIRQPPGKGLEWIGLIWGSGDTYYN SPLKSRLTISKDTSKSQVSLKLSSVTAADT AVYYCAKQTNLWAYDLYSMDYWGQGTLVTV SSASTKGPQVQLVESGGGVVQPGRSLRLSC TASGFTFSMFGVHWVRQAPGKGLEWVAAVS YDGSNKYYAESVKGRFTISRDNSKNILFLQ MDSLRLEDTAVYYCARGRPKVVIPAPLAHW GQGTLVTFSS 1B12.34. SEQ ID NO: DIQMTQSPSSLSASVGDRVTITCQASQDID SS.X3.VL 49 DDLNWYQQKPGKAPKLLISLASILRPGVPS RFSGSGSGTDFTFTISSLQPEDFATYYCLQ SDSFPLTFGQGTKLEIKRTVAAPDIQMTQS PSSVSASVGDRVTITCRASQGISSWLAWYQ QKPGKAPKLLIYEASNLETGVPSRFSGSGS GSDFTLTISSLQPEDFATYYCQQTSSFLLS FGGGTKVEHKR 1B12.A1. SEQ ID NO: EVQLQESGPGLVKPSETLSLTCTVSGFSLR SS.X3.VH 50 DYGVSWIRQPPGKGLEWLGLIWGSGDTYYN SPLKSRLTISKDTSKSQVSLKLSSVTAADT AVYYCAKQTNIWGYDLYGMDYWGQGTLVTV SSASTKGPQVQLVESGGGVVQPGRSLRLSC TASGFTFSMFGVHWVRQAPGKGLEWVAAVS YDGSNKYYAESVKGRFTISRDNSKNILFLQ MDSLRLEDTAVYYCARGRPKVVIPAPLAHW GQGTLVTFSS 1B12.A1. SEQ ID NO: DIQMTQSPSSLSASVGDRVTITCQASQDID SS.X3.VL 51 MDLNWYQQKPGKAPKLLISQANTLPPGVPS RFSGSGSGTDFTFTISSLQPEDFATYYCLQ SDWLPLTFGQGTKLEIKRTVAAPDIQMTQS PSSVSASVGDRVTITCRASQGISSWLAWYQ QKPGKAPKLLIYEASNLETGVPSRFSGSGS GSDFTLTISSLQPEDFATYYCQQTSSFLLS FGGGTKVEHKR 1B12.A3. SEQ ID NO: EVQLQESGPGLVKPSETLSLTCTVSGFSLS SS.X3.VH 52 DYGVSWIRQPPGKGLEWIGLIWGGGDTYYN SPLKSRLTISKDNSKSQVSLKLSSVTAADT AVYYCARQTNLWAYDLYSMDYWGQGTLVTV SSASTKGPQVQLVESGGGVVQPGRSLRLSC TASGFTFSMFGVHWVRQAPGKGLEWVAAVS YDGSNKYYAESVKGRFTISRDNSKNILFLQ MDSLRLEDTAVYYCARGRPKVVIPAPLAHW GQGTLVTFSS 1B12.A3. SEQ ID NO: DIQMTQSPSSLSASVGDRVTITCQASTDID SS.X3.VL 53 DDLNWYQQKPGKAPKLLISLGSTLRPGVPS RFSGSGSGTDFTFTISSLQPEDFATYYCLQ SDRLPLTFGQGTKLEIKRTVAAPDIQMTQS PSSVSASVGDRVTITCRASQGISSWLAWYQ QKPGKAPKLLIYEASNLETGVPSRFSGSGS GSDFTLTISSLQPEDFATYYCQQTSSFLLS FGGGTKVEHKR 1B12.1- SEQ ID NO: EVQLQESGPGLVKPSETLSLTCTVSGFSLS SS-X3 VH 54 DYGVSWIRQPPGKGLEWLGLIWGGGDTYYN SPLKSRLTISKDNSKSQVSLKLSSVTAADT AVYYCAKQRTLWGYDLYGMDYWGQGTLVTV SSASTKGPQVQLVESGGGVVQPGRSLRLSC TASGFTFSMFGVHWVRQAPGKGLEWVAAVS YDGSNKYYAESVKGRFTISRDNSKNILFLQ MDSLRLEDTAVYYCARGRPKVVIPAPLAHW GQGTLVTFSS 1B12.1- SEQ ID NO: DTQVTQSPSSLSASVGDRVTITCITSTDID SS-X3 VL 55 VDMNWYQQKPGKPPKLLISQGNTLRPGVPS RFSSSGSGTDFTFTISSLQPEDFATYYCLQ SDNLPLTFGQGTKLEIKRTVAAPDIQMTQS PSSVSASVGDRVTITCRASQGISSWLAWYQ QKPGKAPKLLIYEASNLETGVPSRFSGSGS GSDFTLTISSLQPEDFATYYCQQTSSFLLS FGGGTKVEHKR

“Dual variable domain” (“DVD”) binding proteins of the disclosure (see, e.g., Table 4 above) comprise two or more antigen binding sites and are tetravalent or multivalent binding proteins. DVDs may be monospecific, i.e., capable of binding one antigen, or multispecific, i.e., capable of binding two or more antigens. A DVD binding protein comprising two heavy chain DVD polypeptides and two light chain DVD polypeptides is referred to as a “DVD immunoglobulin” or “DVD-Ig”. Each half of a DVD-Ig comprises a heavy chain DVD polypeptide and a light chain DVD polypeptide, and two or more antigen binding sites. Each binding site comprises a heavy chain variable domain and a light chain variable domain with a total of six CDRs involved in antigen binding per antigen binding site. A description of the design, expression, and characterization of DVD-Ig molecules is provided in PCT Publication No. WO 2007/024715; U.S. Pat. No. 7,612,181; and Wu et al., Nature Biotechnol., 25: 1290-1297 (2007). A preferred example of such DVD-Ig molecules comprises a heavy chain that comprises the structural formula VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker with the proviso that it is not CH1, X2 is an Fc region, and n is 0 or 1, but preferably 1; and a light chain that comprises the structural formula VD1-(X1)n-VD2-C-(X2)n, wherein VD 1 is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker with the proviso that it is not CH1, and X2 does not comprise an Fc region; and n is 0 or 1, but preferably 1. Such a DVD-Ig may comprise two such heavy chains and two such light chains, wherein each chain comprises variable domains linked in tandem without an intervening constant region between variable regions, wherein a heavy chain and a light chain associate to form tandem functional antigen binding sites, and a pair of heavy and light chains may associate with another pair of heavy and light chains to form a tetrameric binding protein with four functional antigen binding sites. In another example, a DVD-Ig molecule may comprise heavy and light chains that each comprise three variable domains (VD1, VD2, VD3) linked in tandem without an intervening constant region between variable domains, wherein a pair of heavy and light chains may associate to form three antigen binding sites, and wherein a pair of heavy and light chains may associate with another pair of heavy and light chains to form a tetrameric binding protein with six antigen binding sites.

A DVD-Ig binding protein may bind one or more epitopes of IL-1α and/or β. A DVD-Ig binding protein may also bind an epitope of IL-1β and an epitope of a second target antigen other than an IL-1β polypeptide.

The disclosure also provides nucleic acid molecules that encode the binding proteins described herein. For example, the disclosure provides nucleic acid molecule that encode binding proteins comprising any one or more of the CDRs described above. The sequences of these CDRs include SEQ ID NOs:7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 and 29. The disclosure also provides nucleic acid molecules that encode any of the VH or VL domains described herein. Examples of these nucleic acid molecules are provided in Table 5, below.

TABLE 5 Nucleic Acid Molecules Encoding VH and VL Sequence Sequence Encodes Identifier 123456789012345678901234567890 1B12.9 VH SEQ ID NO: GAGGTACAACTTCAAGAATCTGGTCCAGGG (SEQ ID 56 CTTGTGAAGCCTAGTGAAACACTGTCACTG NO: 35) ACTTGTACCGTGTCCGGATTTAGCCTGTCC GACTACGGAGTTTCATGGATTCGACAGCCC CCCGGGAAAGGTCTGGAGTGGCTCGGGCTG ATCTGGGGTGGTGGAGACACATATTACAAT TCCCCTCTGAAATCCAGGCTTACCATCAGC AAGGATAACTCAAAAAGCCAAGTTTCATTG AAGCTGAGTTCCGTGACTGCCGCCGACACG GCGGTCTATTATTGTGCAAAACAGAGGACC CTGTGGGGATATGACCTCTACGGAATGGAC TATTGGGGCCAAGGCACACTTGTGACCGTG TCCAGT 1B12.9 VL SEQ ID NO: GACACTCAAATGACACAGTCCCCATCATCT (SEQ ID 57 TTGTCCGCGAGTGTTGGGGACAGAGTAACT NO: 41) ATTACTTGCATAACCAGCACTGATATAGAC GTGGACATGAACTGGTATCAGCAGAAGCCT GGCAAGCCGCCAAAACTGCTTATCTCTCAA GGCAATACCCTGCGACCCGGCGTGCCTTCT AGATTCAGCTCTAGCGGAAGCGGAACCGAT TTTACCTTCACTATCTCAAGCCTCCAGCCC GAAGACTTCGCCACCTACTATTGTCTGCAG AGCGATAACCTCCCCTTGACTTTCGGGCAA GGTACGAAGCTTGAGATCAAGCGT

The disclosure also provides nucleic acid molecules that encode binding proteins that include two or more VH or VL domains. Examples of these proteins are shown above in Table 4. Examples of nucleic acid molecules that encode these proteins provided in Table 6, below.

TABLE 6 Nucleic Acid Molecules Binding Proteins Sequence Sequence Encodes Identifier 123456789012345678901234567890 1B12.13.SS.X3.VH SEQ ID NO: 58 GAGGTACAACTTCAAGAATCTGGTCCAGGG (SEQ ID NO: 44) CTTGTGAAGCCTAGTGAAACACTGTCACTG ACTTGTACCGTGTCCGGATTTAGCCTGAGC GACTACGGAGTTTCATGGATTCGACAGCCT CCAGGGAAAGGTCTGGAGTGGATCGGGCTG ATCTGGGGCAGCGGAGATACATACTACAAT TCCCCTCTGAAATCCAGGCTTACCATCAGC AAGGATAACTCAAAAAGCCAAGTTTCATTG AAGCTGAGTTCCGTGACTGCCGCCGACACG GCGGTCTATTATTGTGCAAAACAGACGAAC ATCTGGGCGTACGACCTCTACTCCATGGAC TATTGGGGCCAAGGCACACTTGTGACCGTG TCCAGTGCTAGCACTAAAGGACCGCAGGTG CAGCTGGTGGAAAGCGGCGGCGGCGTGGTG CAGCCGGGCCGCAGCCTGCGCCTGAGCTGC ACCGCGAGCGGCTTTACCTTTAGCATGTTT GGCGTGCATTGGGTGCGCCAGGCGCCGGGC AAAGGCCTGGAATGGGTGGCGGCGGTGAGC TATGATGGCAGCAACAAATATTATGCGGAA AGCGTGAAAGGCCGCTTTACCATTAGCCGC GATAACAGCAAAAACATTCTGTTTCTGCAG ATGGATAGCCTGCGCCTGGAAGATACCGCG GTGTATTATTGCGCGCGCGGCCGCCCGAAA GTGGTGATTCCGGCGCCGCTGGCGCATTGG GGCCAGGGCACCCTGGTGACCTTTAGCAGC 1B12.13.SS.X3.VL SEQ ID NO: 59 GACATTCAAATGACACAGTCCCCATCATCT (SEQ ID NO: 45) TTGTCCGCGAGTGTTGGGGACAGAGTAACT ATTACTTGCCAAACCAGTACTGATATAGAC GACGACCTGAACTGGTATCAGCAGAAGCCT GGCAAGGCGCCAAAACTGCTTATCTCTCTC GCCAGTACGCTGCGCCCGGGCGTGCCTTCT AGATTCAGCGGCAGCGGAAGCGGAACCGAT TTTACCTTCACTATCTCAAGCCTCCAGCCC GAAGACTTCGCCACCTACTATTGTCTGCAG AGTGATAGGTTGCCCTTGACTTTCGGGCAA GGTACGAAGCTTGAGATCAAGCGTACCGTC GCAGCTCCTGATATTCAGATGACCCAGAGC CCGAGCAGCGTGAGCGCGAGCGTGGGCGAT CGCGTGACCATTACCTGCCGCGCGAGCCAG GGCATTAGCAGCTGGCTGGCGTGGTATCAG CAGAAACCGGGCAAAGCGCCGAAACTGCTG ATTTATGAAGCGAGCAACCTGGAAACCGGC GTGCCGAGCCGCTTTAGCGGCAGCGGCAGC GGCAGCGATTTTACCCTGACCATTAGCAGC CTGCAGCCGGAAGATTTTGCGACCTATTAT TGCCAGCAGACCAGCAGCTTTCTGCTGAGC TTTGGCGGCGGCACCAAAGTGGAACATAAA CGC 1B12.21.SS.X3.VH SEQ ID NO: 60 GAGGTACAACTTCAAGAATCTGGTCCAGGG (SEQ ID NO: 46) CTTGTGAAGCCTAGTGAAACACTGTCACTG ACTTGTACCGTGTCCGGATTTAGCCTGAGC GAGTTCGGAGTTTCATGGATTCGACAGCCT CCAGGGAAAGGTCTGGAGTGGATCGGGCTG ATCTGGGGCGGGGGAGACACATACTACAAT TCCCCTCTGAAATCCAGGCTTACCATCAGC AAGGATAACTCAAAAAGCCAAGTTTCATTG AAGCTGAGTTCCGTGACTGCCGCCGACACG GCGGTCTATTATTGTGCAAAACAGAGGAAC CTGTGGGCGTACGATTTGTACGGCATGGAC TATTGGGGCCAAGGCACACTTGTGACCGTG TCCAGTGCTAGCACTAAAGGACCGCAGGTG CAGCTGGTGGAAAGCGGCGGCGGCGTGGTG CAGCCGGGCCGCAGCCTGCGCCTGAGCTGC ACCGCGAGCGGCTTTACCTTTAGCATGTTT GGCGTGCATTGGGTGCGCCAGGCGCCGGGC AAAGGCCTGGAATGGGTGGCGGCGGTGAGC TATGATGGCAGCAACAAATATTATGCGGAA AGCGTGAAAGGCCGCTTTACCATTAGCCGC GATAACAGCAAAAACATTCTGTTTCTGCAG ATGGATAGCCTGCGCCTGGAAGATACCGCG GTGTATTATTGCGCGCGCGGCCGCCCGAAA GTGGTGATTCCGGCGCCGCTGGCGCATTGG GGCCAGGGCACCCTGGTGACCTTTAGCAGC 1B12.21.SS.X3.VL SEQ ID NO: 61 GACATTCAAATGACACAGTCCCCATCATCT (SEQ ID NO: 47) TTGTCCGCGAGTGTTGGGGACAGAGTAACT ATTACTTGCCAAACCAGCCAAGATATAGAC ATGGACCTGAACTGGTATCAGCAGAAGCCT GGCAAGGCGCCAAAACTGCTTATCTCTCAG GGCAGTACGCTGTGGCCGGGCGTGCCTTCT AGATTCAGCGGCAGCGGAAGCGGAACCGAT TTTACCTTCACTATCTCAAGCCTCCAGCCC GAAGACTTCGCCACCTACTATTGTCTGCAG ACGGATAGTTTCCCCTTGACTTTCGGGCAA GGTACGAAGCTTGAGATCAAGCGTACCGTC GCAGCTCCTGATATTCAGATGACCCAGAGC CCGAGCAGCGTGAGCGCGAGCGTGGGCGAT CGCGTGACCATTACCTGCCGCGCGAGCCAG GGCATTAGCAGCTGGCTGGCGTGGTATCAG CAGAAACCGGGCAAAGCGCCGAAACTGCTG ATTTATGAAGCGAGCAACCTGGAAACCGGC GTGCCGAGCCGCTTTAGCGGCAGCGGCAGC GGCAGCGATTTTACCCTGACCATTAGCAGC CTGCAGCCGGAAGATTTTGCGACCTATTAT TGCCAGCAGACCAGCAGCTTTCTGCTGAGC TTTGGCGGCGGCACCAAAGTGGAACATAAA CGC 1B12. 34.SS.X3.VH SEQ ID NO: 62 GAGGTACAACTTCAAGAATCTGGTCCAGGG (SEQ ID NO: 48) CTTGTGAAGCCTAGTGAAACACTGTCACTG ACTTGTACCGTGTCCGGATTTAGCCTGAGC GACTACGGAGTTTCATGGATTCGACAGCCT CCAGGGAAAGGTCTGGAGTGGATCGGGCTG ATCTGGGGGTCGGGAGATACATACTACAAT TCCCCTCTGAAATCCAGGCTTACCATCAGC AAGGATACCTCAAAAAGCCAAGTTTCATTG AAGCTGAGTTCCGTGACTGCCGCCGACACG GCGGTCTATTATTGTGCAAAACAGACGAAC CTGTGGGCGTACGACCTCTACAGCATGGAC TATTGGGGCCAAGGCACACTTGTGACCGTG TCCAGTGCTAGCACTAAAGGACCGCAGGTG CAGCTGGTGGAAAGCGGCGGCGGCGTGGTG CAGCCGGGCCGCAGCCTGCGCCTGAGCTGC ACCGCGAGCGGCTTTACCTTTAGCATGTTT GGCGTGCATTGGGTGCGCCAGGCGCCGGGC AAAGGCCTGGAATGGGTGGCGGCGGTGAGC TATGATGGCAGCAACAAATATTATGCGGAA AGCGTGAAAGGCCGCTTTACCATTAGCCGC GATAACAGCAAAAACATTCTGTTTCTGCAG ATGGATAGCCTGCGCCTGGAAGATACCGCG GTGTATTATTGCGCGCGCGGCCGCCCGAAA GTGGTGATTCCGGCGCCGCTGGCGCATTGG GGCCAGGGCACCCTGGTGACCTTTAGCAGC 1B12.34.SS.X3.VL SEQ ID NO: 63 GACATTCAAATGACACAGTCCCCATCATCT (SEQ ID NO: 49) TTGTCCGCGAGTGTTGGGGACAGAGTAACT ATTACTTGCCAAGCCAGCCAAGATATAGAC GACGACCTGAACTGGTATCAGCAGAAGCCT GGCAAGGCGCCAAAACTGCTTATCTCTCTG GCCAGTATCCTGCGCCCGGGCGTGCCTTCT AGATTCAGCGGCAGCGGAAGCGGAACCGAT TTTACCTTCACTATCTCAAGCCTCCAGCCC GAAGACTTCGCCACCTACTATTGTCTGCAG AGTGACAGTTTCCCCTTGACTTTCGGGCAA GGTACGAAGCTTGAGATCAAGCGTACCGTC GCAGCTCCTGATATTCAGATGACCCAGAGC CCGAGCAGCGTGAGCGCGAGCGTGGGCGAT CGCGTGACCATTACCTGCCGCGCGAGCCAG GGCATTAGCAGCTGGCTGGCGTGGTATCAG CAGAAACCGGGCAAAGCGCCGAAACTGCTG ATTTATGAAGCGAGCAACCTGGAAACCGGC GTGCCGAGCCGCTTTAGCGGCAGCGGCAGC GGCAGCGATTTTACCCTGACCATTAGCAGC CTGCAGCCGGAAGATTTTGCGACCTATTAT TGCCAGCAGACCAGCAGCTTTCTGCTGAGC TTTGGCGGCGGCACCAAAGTGGAACATAAA CGC 1B12.A1.SS.X3.VH SEQ ID NO: 64 GAGGTACAACTTCAAGAATCTGGTCCAGGG (SEQ ID NO: 50) CTTGTGAAGCCTAGTGAAACACTGTCACTG ACTTGTACCGTGTCCGGATTTAGCCTGAGG GACTACGGAGTTTCATGGATTCGACAGCCT CCAGGGAAAGGTCTGGAGTGGCTCGGGCTG ATCTGGGGGAGCGGAGACACATACTACAAT TCCCCTCTGAAATCCAGGCTTACCATCAGC AAGGATACCTCAAAAAGCCAAGTTTCATTG AAGCTGAGTTCCGTGACTGCCGCCGACACG GCGGTCTATTATTGTGCAAAACAGACGAAC ATCTGGGGCTACGATCTCTACGGCATGGAC TATTGGGGCCAAGGCACACTTGTGACCGTG TCCAGTGCTAGCACTAAAGGACCGCAGGTG CAGCTGGTGGAAAGCGGCGGCGGCGTGGTG CAGCCGGGCCGCAGCCTGCGCCTGAGCTGC ACCGCGAGCGGCTTTACCTTTAGCATGTTT GGCGTGCATTGGGTGCGCCAGGCGCCGGGC AAAGGCCTGGAATGGGTGGCGGCGGTGAGC TATGATGGCAGCAACAAATATTATGCGGAA AGCGTGAAAGGCCGCTTTACCATTAGCCGC GATAACAGCAAAAACATTCTGTTTCTGCAG ATGGATAGCCTGCGCCTGGAAGATACCGCG GTGTATTATTGCGCGCGCGGCCGCCCGAAA GTGGTGATTCCGGCGCCGCTGGCGCATTGG GGCCAGGGCACCCTGGTGACCTTTAGCAGC 1B12.A1.SS.X3.VL SEQ ID NO: 65 GACATTCAAATGACACAGTCCCCATCATCT (SEQ ID NO: 51) TTGTCCGCGAGTGTTGGGGACAGAGTAACT ATTACTTGCCAAGCCAGCCAAGATATAGAC ATGGACCTGAACTGGTATCAGCAGAAGCCT GGCAAGGCGCCAAAACTGCTTATCTCTCAG GCCAATACGCTGCCCCCGGGCGTGCCTTCT AGATTCAGCGGCAGCGGAAGCGGAACCGAT TTTACCTTCACTATCTCAAGCCTCCAGCCC GAAGACTTCGCCACCTACTATTGTCTGCAG AGTGATTGGTTGCCCTTGACTTTCGGGCAA GGTACGAAGCTTGAGATCAAGCGTACCGTC GCAGCTCCTGATATTCAGATGACCCAGAGC CCGAGCAGCGTGAGCGCGAGCGTGGGCGAT CGCGTGACCATTACCTGCCGCGCGAGCCAG GGCATTAGCAGCTGGCTGGCGTGGTATCAG CAGAAACCGGGCAAAGCGCCGAAACTGCTG ATTTATGAAGCGAGCAACCTGGAAACCGGC GTGCCGAGCCGCTTTAGCGGCAGCGGCAGC GGCAGCGATTTTACCCTGACCATTAGCAGC CTGCAGCCGGAAGATTTTGCGACCTATTAT TGCCAGCAGACCAGCAGCTTTCTGCTGAGC TTTGGCGGCGGCACCAAAGTGGAACATAAA CGC 1B12.A3.SS.X3.VH SEQ ID NO: 66 GAGGTACAACTTCAAGAATCTGGTCCAGGG (SEQ ID NO: 52) CTTGTGAAGCCTAGTGAAACACTGTCACTG ACTTGTACCGTGTCCGGATTTAGCCTGAGC GACTACGGAGTTTCATGGATTCGACAGCCT CCAGGGAAAGGTCTGGAGTGGATCGGGCTC ATCTGGGGCGGGGGAGATACATACTACAAT TCCCCTCTGAAATCCAGGCTTACCATCAGC AAGGATAACTCAAAAAGCCAAGTTTCATTG AAGCTGAGTTCCGTGACTGCCGCCGACACG GCGGTCTATTATTGTGCAAGACAGACGAAC CTGTGGGCGTACGACTTGTACAGCATGGAC TATTGGGGCCAAGGCACACTTGTGACCGTG TCCAGTGCTAGCACTAAAGGACCGCAGGTG CAGCTGGTGGAAAGCGGCGGCGGCGTGGTG CAGCCGGGCCGCAGCCTGCGCCTGAGCTGC ACCGCGAGCGGCTTTACCTTTAGCATGTTT GGCGTGCATTGGGTGCGCCAGGCGCCGGGC AAAGGCCTGGAATGGGTGGCGGCGGTGAGC TATGATGGCAGCAACAAATATTATGCGGAA AGCGTGAAAGGCCGCTTTACCATTAGCCGC GATAACAGCAAAAACATTCTGTTTCTGCAG ATGGATAGCCTGCGCCTGGAAGATACCGCG GTGTATTATTGCGCGCGCGGCCGCCCGAAA GTGGTGATTCCGGCGCCGCTGGCGCATTGG GGCCAGGGCACCCTGGTGACCTTTAGCAGC 1B12.A3.SS.X3.VL SEQ ID NO: 67 GACATTCAAATGACACAGTCCCCATCATCT (SEQ ID NO: 53) TTGTCCGCGAGTGTTGGGGACAGAGTAACT ATTACTTGCCAAGCCAGCACTGATATAGAC GACGACCTGAACTGGTATCAGCAGAAGCCT GGCAAGGCGCCAAAACTGCTTATCTCTCTG GGCAGTACCCTGCGGCCCGGCGTGCCTTCT AGATTCAGCGGCAGCGGAAGCGGAACCGAT TTTACCTTCACTATCTCAAGCCTCCAGCCC GAAGACTTCGCCACCTACTATTGTCTGCAG AGTGATAGGTTGCCCTTGACTTTCGGGCAA GGTACGAAGCTTGAGATCAAGCGTACCGTC GCAGCTCCTGATATTCAGATGACCCAGAGC CCGAGCAGCGTGAGCGCGAGCGTGGGCGAT CGCGTGACCATTACCTGCCGCGCGAGCCAG GGCATTAGCAGCTGGCTGGCGTGGTATCAG CAGAAACCGGGCAAAGCGCCGAAACTGCTG ATTTATGAAGCGAGCAACCTGGAAACCGGC GTGCCGAGCCGCTTTAGCGGCAGCGGCAGC GGCAGCGATTTTACCCTGACCATTAGCAGC CTGCAGCCGGAAGATTTTGCGACCTATTAT TGCCAGCAGACCAGCAGCTTTCTGCTGAGC TTTGGCGGCGGCACCAAAGTGGAACATAAA CGC 1B12.1-SS-X3 VH (SEQ SEQ ID NO: 68 GAGGTACAACTTCAAGAATCTGGTCCAGGG ID NO: 54) CTTGTGAAGCCTAGTGAAACACTGTCACTG ACTTGTACCGTGTCCGGATTTAGCCTGTCC GACTACGGAGTTTCATGGATTCGACAGCCC CCCGGGAAAGGTCTGGAGTGGCTCGGGCTG ATCTGGGGTGGTGGAGACACATATTACAAT TCCCCTCTGAAATCCAGGCTTACCATCAGC AAGGATAACTCAAAAAGCCAAGTTTCATTG AAGCTGAGTTCCGTGACTGCCGCCGACACG GCGGTCTATTATTGTGCAAAACAGAGGACC CTGTGGGGATATGACCTCTACGGAATGGAC TATTGGGGCCAAGGCACACTTGTGACCGTG TCCAGTGCCTCAACAAAAGGGCCTCAGGTG CAGCTGGTGGAAAGCGGCGGCGGCGTGGTG CAGCCGGGCCGCAGCCTGCGCCTGAGCTGC ACCGCGAGCGGCTTTACCTTTAGCATGTTT GGCGTGCATTGGGTGCGCCAGGCGCCGGGC AAAGGCCTGGAATGGGTGGCGGCGGTGAGC TATGATGGCAGCAACAAATATTATGCGGAA AGCGTGAAAGGCCGCTTTACCATTAGCCGC GATAACAGCAAAAACATTCTGTTTCTGCAG ATGGATAGCCTGCGCCTGGAAGATACCGCG GTGTATTATTGCGCGCGCGGCCGCCCGAAA GTGGTGATTCCGGCGCCGCTGGCGCATTGG GGCCAGGGCACCCTGGTGACCTTTAGCAGC 1B12.1-SS-X3 VL (SEQ SEQ ID NO: 69 GACACTCAAGTGACACAGTCCCCATCATCT ID NO: 55) TTGTCCGCGAGTGTTGGGGACAGAGTAACT ATTACTTGCATAACCAGCACTGATATAGAC GTGGACATGAACTGGTATCAGCAGAAGCCT GGCAAGCCGCCAAAACTGCTTATCTCTCAA GGCAATACCCTGCGACCCGGCGTGCCTTCT AGATTCAGCTCTAGCGGAAGCGGAACCGAT TTTACCTTCACTATCTCAAGCCTCCAGCCC GAAGACTTCGCCACCTACTATTGTCTGCAG AGCGATAACCTCCCCTTGACTTTCGGGCAA GGTACGAAGCTTGAGATCAAGCGTACGGTG GCTGCACCAGATATTCAGATGACCCAGAGC CCGAGCAGCGTGAGCGCGAGCGTGGGCGAT CGCGTGACCATTACCTGCCGCGCGAGCCAG GGCATTAGCAGCTGGCTGGCGTGGTATCAG CAGAAACCGGGCAAAGCGCCGAAACTGCTG ATTTATGAAGCGAGCAACCTGGAAACCGGC GTGCCGAGCCGCTTTAGCGGCAGCGGCAGC GGCAGCGATTTTACCCTGACCATTAGCAGC CTGCAGCCGGAAGATTTTGCGACCTATTAT TGCCAGCAGACCAGCAGCTTTCTGCTGAGC TTTGGCGGCGGCACCAAAGTGGAACATAAA CGC

The disclosure also provides nucleic acid molecules that are 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to a nucleic acid molecule that can encode any of the CDRs, VH, VL or any other binding protein described herein. The disclosure also provides fragments of these nucleic acid molecules. In certain embodiments, fragments can have 363 nucleic acids or fewer. In other embodiments, fragments can have 321 nucleic acids or fewer. In other embodiments, fragments can have 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 195, 210, 225, 240, 255, 270, 285, 300, 315 or fewer nucleic acids.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of the term “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

Abbreviations used herein include Ag, meaning antigen; DNA, meaning deoxyribonucleic acid; FACS, meaning fluorescence-activated cell sorting; IgG, meaning immunoglobulin G; PCR, meaning polymerase chain reaction; PE, meaning R-Phycoerythrin; ScFv, meaning single chain variable fragment; CDR, meaning complementarity determining region; Cyno, meaning cynomolgus; MACS, meaning magnetic activated cell sorting; VH, meaning variable heavy and Vk, meaning variable kappa.

Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

That the present disclosure may be more readily understood, select terms are defined below.

The term “polypeptide” refers to any polymeric chain of amino acids. The terms “peptide” and “protein” are used interchangeably with the term polypeptide and also refer to a polymeric chain of amino acids. The term “polypeptide” encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. A polypeptide may be monomeric or polymeric. The term “polypeptide” encompasses fragments and variants (including fragments of variants) thereof, unless otherwise contradicted by context. For an antigenic polypeptide, a fragment of polypeptide optionally contains at least one contiguous or nonlinear epitope of polypeptide. The precise boundaries of the at least one epitope fragment can be confirmed using ordinary skill in the art. The fragment comprises at least about 5 contiguous amino acids, such as at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, or at least about 20 contiguous amino acids. A variant of polypeptide is as described herein.

The term “isolated protein” or “isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally associated components that accompany it in its native state; is substantially free of other proteins from the same species; is expressed by a cell from a different species; or does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.

The term “recovering” refers to the process of rendering a chemical species such as a polypeptide substantially free of naturally associated components by isolation, e.g., using protein purification techniques well known in the art.

The term “human IL-1α” (abbreviated herein as hIL-1α, or IL-1 α) includes a pleiotropic cytokine involved in various immune responses, inflammatory processes, and hematopoiesis. For example, IL-1α includes the human cytokine produced by activated macrophages; it stimulates thymocyte proliferation by inducing IL-2 release, B-cell maturation and proliferation, and fibroblast growth factor activity. The term human IL-1α is intended to include recombinant human IL-1α (rh IL-1α) that can be prepared by standard recombinant expression methods.

The term “human IL-1β” (abbreviated herein as hIL-1β, or IL-1β) includes a pleiotropic cytokine involved in various immune responses, inflammatory processes, and hematopoiesis. The term human IL-1β includes recombinant human IL-1β (rh IL-1β) that can be prepared by standard recombinant expression methods.

The amino acid sequences of human IL-1α and IL-1β are shown in Table 7.

TABLE 7 Sequence of Human IL-1α and Human IL-1β Sequence Sequence Protein Identifier 123456789012345678901234567890 Mature SEQ ID NO: SAPFSFLSNVKYNFMRIIKYEFILNDALNQ human 70 SIIRANDQYLTAAALHNLDEAVKFDMGAYK IL-1α SSKDDAKITVILRISKTQLYVTAQDEDQPV LLKEMPEIPKTITGSETNLLFFWETHGTKN YFTSVAHPNLFIATKQDYWVCLAGGPPSIT DFQILENQA Mature SEQ ID NO: APVRSLNCTLRDSQQKSLVMSGPYELKALH human 71 LQGQDMEQQVVFSMSFVQGEESNDKIPVAL IL-1β GLKEKNLYLSCVLKDDKPTLQLESVDPKNY PKKKMEKRFVFNKIEINNKLEFESAQFPNW YISTSQAENMPVFLGGTKGGQDITDFTMQF VSS

The term “biological activity” refers to all inherent biological properties of the IL-1 cytokine, e.g., IL-1α and/or IL-1β. Biological properties of IL-1α and IL-1β include, but are not limited to, binding to an IL-1 receptor.

The terms “specific binding” or “specifically binding” in reference to the interaction of an antibody, a binding protein, or a peptide with a second chemical species, mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody. In certain embodiments, a binding protein that specifically binds to an antigen binds to that antigen with a K_(D) greater than 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³ or 10⁻¹⁴ M. In other embodiments, a binding protein that specifically binds to an antigen binds to that antigen with a K_(D) of between 10⁻⁶ and 10⁻⁷, 10⁻⁶ and 10⁻⁸, 10⁻⁶ and 10⁻⁹, 10⁻⁶ and 10⁻¹⁰, 10⁻⁶ and 10⁻¹¹, 10⁻⁶ and 10⁻¹², 10⁻⁶ and 10⁻¹³, 10⁻⁶ and 10⁻¹⁴, 10⁻⁹ and 10⁻¹⁰, 10⁻⁹ and 10⁻¹¹, 10⁻⁹ and 10⁻¹², 10⁻⁹ and 10⁻¹³ or 10⁻⁹ and 10⁻¹⁴M.

The term “antibody” broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art. Nonlimiting embodiments of which are discussed below.

In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains: CH1, CH2, and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgA1 and IgA2) or subclass.

The term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, which may be generated by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain, and a CH3 domain, and optionally comprises a CH4 domain. Replacements of amino acid residues in the Fc portion to alter antibody effector function are known in the art (Winter et al., U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of an antibody mediates several important effector functions, for example, cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC), and half-life/clearance rate of antibody and antigen-antibody complexes. In some cases these effector functions are desirable for therapeutic antibody but in other cases might be unnecessary or even deleterious, depending on the therapeutic objectives. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγRs and complement C1q, respectively. Neonatal Fc receptors (FcRn) are the critical components determining the circulating half-life of antibodies. In still another embodiment at least one amino acid residue is replaced in the constant region of the antibody, for example the Fc region of the antibody, such that effector functions of the antibody are altered. The dimerization of two identical heavy chains of an immunoglobulin is mediated by the dimerization of CH3 domains and is stabilized by the disulfide bonds within the hinge region (Huber et al., Nature, 264: 415-420 (1976); Thies et al., J. Mol. Biol., 293: 67-79 (1999)). Mutation of cysteine residues within the hinge regions to prevent heavy chain-heavy chain disulfide bonds will destabilize dimerization of CH3 domains. In certain embodiments, residues 234 and 235 of the IgG1 Fc region are mutated from LL to AA (Alegre M L, Peterson, L J, Xu, D., et al. Transplantation 1994; 57:1537; Armour, K L., van de Winkel, J. G. J., et al. 2003; Molecular Immunology; 40:585.) Other residues responsible for CH3 dimerization have been identified (Dall'Acqua et al., Biochemistry, 37: 9266-9273 (1998)). Therefore, it is possible to generate a monovalent half-Ig. Interestingly, these monovalent half Ig molecules have been found in nature for both IgG and IgA subclasses (Seligmann et al., Ann. Immunol., 129 C: 855-870 (1978); Biewenga et al., Clin. Exp. Immunol., 51: 395-400 (1983)). The stoichiometry of FcRn: Ig Fc region has been determined to be 2:1 (West et al., Biochemistry, 39: 9698-9708 (2000)), and half Fc is sufficient for mediating FcRn binding (Kim et al., Eur. J. Immunol., 24: 542-548 (1994)). Mutations to disrupt the dimerization of CH3 domain may not have greater adverse effect on its FcRn binding as the residues important for CH3 dimerization are located on the inner interface of CH3 b sheet structure, whereas the region responsible for FcRn binding is located on the outside interface of CH2-CH3 domains. However, the half Ig molecule may have certain advantage in tissue penetration due to its smaller size than that of a regular antibody. In one embodiment, at least one amino acid residue is replaced in the constant region of the binding protein of the disclosure, for example the Fc region, such that the dimerization of the heavy chains is disrupted, resulting in half DVD Ig molecules. The anti-inflammatory activity of IgG is completely dependent on sialylation of the N-linked glycan of the IgG Fc fragment. The precise glycan requirements for anti-inflammatory activity has been determined, such that an appropriate IgG1 Fc fragment can be created, thereby generating a fully recombinant, sialylated IgG1 Fc with greatly enhanced potency (Anthony et al., Science, 320:373-376 (2008)).

The term “antigen-binding portion” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., hIL-1α or hIL-1β). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual specific, or multispecific formats; specifically binding to two or more different antigens (e.g., hIL-1β and a different antigen molecule, such as hIL-1α). Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature, 341: 544-546 (1989); PCT Publication No. WO 90/05144), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al., Science, 242: 423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988)). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993); Poljak, R. J., Structure, 2: 1121-1123 (1994)). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (Springer-Verlag, New York, 2001), p. 790 (ISBN 3-540-41354-5)). In addition single chain antibodies also include “linear antibodies” comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al., Protein Eng., 8(10): 1057-1062 (1995); and U.S. Pat. No. 5,641,870)).

An immunoglobulin constant (C) domain refers to a heavy (CH) or light (CL) chain constant domain. Murine and human IgG heavy chain and light chain constant domain amino acid sequences are known in the art.

The term “IL-1α or β binding protein construct” (or “binding protein construct”) refers to a polypeptide comprising one or more of the antigen binding portions of the disclosure linked to a linker or an immunoglobulin constant domain. A “linker polypeptide” comprises two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Such linker polypeptides are well known in the art (see e.g., Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993); Poljak, R. J., Structure, 2: 1121-1123 (1994)). An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art and represented in Table 8.

TABLE 8 Sequence of Human IgG Heavy Chain Constant Domain and Light Chain Constant Domain Sequence Identi- Sequence Protein fier 123456789012345678901234567890 Ig gamma-1 SEQ ID ASTKGPSVFFLAPSSKSTSGGTAALGCLVK constant NO: 72 DYFPEPVTVSWNSGALTSGVHTFPAVLQSS region GLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ig gamma-1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVK constant NO: 73 DYFPEPVTVSWNSGALTSGVHTFPAVLQSS region GLYSLSSVVTVPSSSLGTQTYICNVNHKPS 234, 235 NTKVDKKVEPKSCDKTHTCPPCPAPEAAGG mutant PSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ig Kappa SEQ ID TVAAPSVFIFPPSDEQLKSGTASVVCLLNN constant NO: 74 FYPREAKVQWKVDNALQSGNSQESVTEQDS region KDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC Ig Lambda SEQ ID QPKAAPSVTLFPPSSEELQANKATLVCLIS constant NO: 75 DFYPGAVTVAWKADSSPVKAGVETTTPSKQ region SNNKYAASSYLSLTPEQWKSHRSYSCQVTH EGSTVEKTVAPTECS

Still further, an IL-1α or β binding protein, an antibody antigen-binding portion thereof, may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody antigen-binding portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov et al., Human Antibod. Hybridomas, 6: 93-101 (1995)) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov et al., Mol. Immunol., 31: 1047-1058 (1994)). Antibody portions, such as Fab and F(ab′)₂ fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antigen-binding portions thereof, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques. An IL-1α or β binding protein, such as an antigen-binding portion of an antibody may also be part of a DVD-Ig™.

An “isolated antibody” is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds hIL-1α or hIL-1β is substantially free of antibodies that specifically bind antigens other than hIL-1α or hIL-1β). An isolated antibody that specifically binds hIL-1α or hIL-1β may, however, have cross-reactivity to other antigens, such as hIL-1α or IL-1β molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

The term “monoclonal antibody” or “mAb” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method.

The term “human antibody” includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody” does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The term “recombinant human antibody” includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further in Section II C, below), antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom, H. R., Trends Biotechnol., 15: 62-70 (1997); Azzazy and Highsmith, Clin. Biochem., 35: 425-445 (2002); Gavilondo and Larrick, BioTechniques, 29: 128-145 (2000); Hoogenboom and Chames, Immunol. Today, 21: 371-378 (2000)), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al., Nucl. Acids Res., 20: 6287-6295 (1992); Kellermann and Green, Curr. Opin. Biotechnol., 13: 593-597 (2002); Little et al., Immunol. Today, 21: 364-370 (2000)); or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

The term “chimeric antibody” refers to antibodies that comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies that comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences.

The term “CDR” refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The term “CDR set” as used herein refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987); and Chothia et al., Nature, 342: 877-883 (1989)) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as L1, L2, and L3 or H1, H2, and H3 where the “L” and the “H” designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan et al. (FASEB J., 9: 133-139 (1995)) and MacCallum et al. (J. Mol. Biol., 262(5): 732-745 (1996)). Still other CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although exemplary embodiments use Kabat or Chothia defined CDRs.

The terms “Kabat numbering”, “Kabat definitions”, and “Kabat labeling” are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al., Ann. NY Acad. Sci., 190: 382-391 (1971); and Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991)). For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.

The growth and analysis of extensive public databases of amino acid sequences of variable heavy and light regions over the past twenty years have led to the understanding of the typical boundaries between framework regions (FR) and CDR sequences within variable region sequences and enabled persons skilled in this art to accurately determine the CDRs according to Kabat numbering, Chothia numbering, or other systems. See, e.g., Martin, “Protein Sequence and Structure Analysis of Antibody Variable Domains,” Chapter 31, In Antibody Engineering, (Kontermann and Dubel, eds.) (Springer-Verlag, Berlin, 2001), especially pages 432-433. A useful method of determining the amino acid sequences of Kabat CDRs within the amino acid sequences of variable heavy (VH) and variable light (VL) regions is provided below:

To identify a CDR-L1 amino acid sequence: Starts approximately 24 amino acid residues from the amino terminus of the VL region; Residue before the CDR-L1 sequence is always cysteine (C); Residue after the CDR-L1 sequence is always a tryptophan (W) residue, typically Trp-Tyr-Gln (W-Y-Q), but also Trp-Leu-Gln (W-L-Q), Trp-Phe-Gln (W-F-Q), and Trp-Tyr-Leu (W-Y-L); Length is typically 10 to 17 amino acid residues.

To identify a CDR-L2 amino acid sequence: Starts always 16 residues after the end of CDR-L1; Residues before the CDR-L2 sequence are generally Ile-Tyr (I-Y), but also Val-Tyr (V-Y), Ile-Lys (1-K), and Ile-Phe (1-F); Length is always 7 amino acid residues.

To identify a CDR-L3 amino acid sequence: Starts always 33 amino acids after the end of CDR-L2; Residue before the CDR-L3 amino acid sequence is always a cysteine (C); Residues after the CDR-L3 sequence are always Phe-Gly-X-Gly (F-G-X-G) (SEQ ID NO:76), where X is any amino acid; Length is typically 7 to 11 amino acid residues.

To identify a CDR-H1 amino acid sequence: Starts approximately 31 amino acid residues from amino terminus of VH region and always 9 residues after a cysteine (C); Residues before the CDR-H1 sequence are always Cys-X-X-X-X-X-X-X-X (SEQ ID NO: 430), where X is any amino acid; Residue after CDR-H1 sequence is always a Trp (W), typically Trp-Val (W-V), but also Trp-Ile (W-I), and Trp-Ala (W-A); Length is typically 5 to 7 amino acid residues.

To identify a CDR-H2 amino acid sequence: Starts always 15 amino acid residues after the end of CDR-H1; Residues before CDR-H2 sequence are typically Leu-Glu-Trp-Ile-Gly (L-E-W-I-G) (SEQ ID NO:77), but other variations also; Residues after CDR-H2 sequence are Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala (K/R-L/I/V/F/T/A-T/S/I/A); Length is typically 16 to 19 amino acid residues.

To identify a CDR-H3 amino acid sequence: Starts always 33 amino acid residues after the end of CDR-H2 and always 3 after a cysteine (C)′ Residues before the CDR-H3 sequence are always Cys-X-X (C-X-X), where X is any amino acid, typically Cys-Ala-Arg (C-A-R); Residues after the CDR-H3 sequence are always Trp-Gly-X-Gly (W-G-X-G) (SEQ ID NO:78), where X is any amino acid; Length is typically 3 to 25 amino acid residues.

As used herein, the term “canonical” residue refers to a residue in a CDR or framework that defines a particular canonical CDR structure as defined by Chothia et al. (J. Mol. Biol., 196: 901-917 (1987)); and Chothia et al. (J. Mol. Biol., 227: 799-817 (1992)), both are incorporated herein by reference). According to Chothia et al., critical portions of the CDRs of many antibodies have nearly identical peptide backbone confirmations despite great diversity at the level of amino acid sequence. Each canonical structure specifies primarily a set of peptide backbone torsion angles for a contiguous segment of amino acid residues forming a loop.

An “affinity matured” antibody is an antibody with one or more alterations in one or more CDRs thereof which result in an improvement in the affinity of the antibody for a target antigen, compared to a parent antibody which does not possess the alteration(s). Exemplary affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. A variety of procedures for producing affinity matured antibodies are known in the art. For example, Marks et al., BioTechnology, 10: 779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91: 3809-3813 (1994); Schier et al., Gene, 169: 147-155 (1995); Yelton et al., J. Immunol., 155: 1994-2004 (1995); Jackson et al., J. Immunol., 154(7): 3310-3319 (1995); Hawkins et al., J. Mol. Biol., 226: 889-896 (1992). Selective mutation at selective mutagenesis positions and at contact or hypermutation positions with an activity enhancing amino acid residue is described in U.S. Pat. No. 6,914,128 B1.

The term “multivalent binding protein” denotes a binding protein comprising two or more antigen binding sites. A multivalent binding protein is preferably engineered to have three or more antigen binding sites, and is generally not a naturally occurring antibody. The term “multispecific binding protein” refers to a binding protein capable of binding two or more related or unrelated targets.

The term “bispecific antibody”, as used herein, refers to full-length antibodies that are generated by quadroma technology (see Milstein and Cuello, Nature, 305: 537-540 (1983)), by chemical conjugation of two different monoclonal antibodies (see Staerz et al., Nature, 314: 628-631 (1985)), or by knob-into-hole or similar approaches which introduces mutations in the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14): 6444-6448 (1993)), resulting in multiple different immunoglobulin species of which only one is the functional bispecific antibody. By molecular function, a bispecific antibody binds one antigen (or epitope) on one of its two binding arms (one pair of HC/LC), and binds a different antigen (or epitope) on its second arm (a different pair of HC/LC). By this definition, a bispecific antibody has two distinct antigen binding arms (in both specificity and CDR sequences), and is monovalent for each antigen it binds.

The term “dual-specific antibody”, as used herein, refers to full-length antibodies that can bind two different antigens (or epitopes) in each of its two binding arms (a pair of HC/LC) (see PCT Publication No. WO 02/02773). Accordingly a dual-specific binding protein has two identical antigen binding arms, with identical specificity and identical CDR sequences, and is bivalent for each antigen to which it binds.

A “functional antigen binding site” of a binding protein is one that is capable of binding a target antigen. The antigen binding affinity of the antigen binding site is not necessarily as strong as the parent antibody from which the antigen binding site is derived, but the ability to bind antigen must be measurable using any one of a variety of methods known for evaluating antibody binding to an antigen. Moreover, the antigen binding affinity of each of the antigen binding sites of a multivalent antibody herein need not be quantitatively the same.

As used herein, the terms “donor” and “donor antibody” refer to an antibody providing one or more CDRs. In an exemplary embodiment, the donor antibody is an antibody from a species different from the antibody from which the framework regions are obtained or derived. In the context of a humanized antibody, the term “donor antibody” refers to a non-human antibody providing one or more CDRs.

As used herein, the term “framework” or “framework sequence” refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, -L2, and -L3 of light chain and CDR-H1, —H2, and —H3 of heavy chain) also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular sub-regions as FR1, FR2, FR3 or FR4, a framework region, as referred by others, represents the combined FR's within the variable region of a single, naturally occurring immunoglobulin chain. As used herein, a FR represents one of the four sub-regions, and FRs represents two or more of the four sub-regions constituting a framework region.

As used herein, the terms “acceptor” and “acceptor antibody” refer to the antibody providing or nucleic acid sequence encoding at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequences of one or more of the framework regions. In some embodiments, the term “acceptor” refers to the antibody amino acid providing or nucleic acid sequence encoding the constant region(s). In yet another embodiment, the term “acceptor” refers to the antibody amino acid providing or nucleic acid sequence encoding one or more of the framework regions and the constant region(s). In a specific embodiment, the term “acceptor” refers to a human antibody amino acid or nucleic acid sequence that provides or encodes at least 80%, preferably, at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequences of one or more of the framework regions. In accordance with this embodiment, an acceptor may contain at least 1, at least 2, at least 3, least 4, at least 5, or at least 10 amino acid residues that does (do) not occur at one or more specific positions of a human antibody. An acceptor framework region and/or acceptor constant region(s) may be, e.g., derived or obtained from a germline antibody gene, a mature antibody gene, a functional antibody (e.g., antibodies well known in the art, antibodies in development, or antibodies commercially available).

Human heavy chain and light chain acceptor sequences are known in the art. In one embodiment of the disclosure the human heavy chain and light chain acceptor sequences are selected from the sequences listed from V-base (http://vbase.mrc-cpe.cam.ac.uk/) or from IMGT®, the international ImMunoGeneTics Information System® (http://imgt.cines.fr/textes/IMGTrepertoire/LocusGenes/). In another embodiment of the disclosure the human heavy chain and light chain acceptor sequences are selected from the sequences described in Table 3 and Table 4 of U.S. Patent Publication No. 2011/0280800, incorporated by reference herein in their entireties.

As used herein, the term “germline antibody gene” or “gene fragment” refers to an immunoglobulin sequence encoded by non-lymphoid cells that have not undergone the maturation process that leads to genetic rearrangement and mutation for expression of a particular immunoglobulin. (See, e.g., Shapiro et al., Crit. Rev. Immunol., 22(3): 183-200 (2002); Marchalonis et al., Adv. Exp. Med. Biol., 484: 13-30 (2001)). One of the advantages provided by various embodiments of the present disclosure stems from the recognition that germline antibody genes are more likely than mature antibody genes to conserve essential amino acid sequence structures characteristic of individuals in the species, hence less likely to be recognized as from a foreign source when used therapeutically in that species.

As used herein, the term “key” residues refer to certain residues within the variable region that have more impact on the binding specificity and/or affinity of an antibody, in particular a humanized antibody. A key residue includes, but is not limited to, one or more of the following: a residue that is adjacent to a CDR, a potential glycosylation site (can be either N- or O-glycosylation site), a rare residue, a residue capable of interacting with the antigen, a residue capable of interacting with a CDR, a canonical residue, a contact residue between heavy chain variable region and light chain variable region, a residue within the Vernier zone, and a residue in the region that overlaps between the Chothia definition of a variable heavy chain CDR/and the Kabat definition of the first heavy chain framework.

The term “humanized antibody” refers to antibodies that comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like”, i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding nonhuman CDR sequences. Also “humanized antibody” is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term “substantially” in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab′, F(aN)₂, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In an embodiment, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody only contains a humanized light chain. In some embodiments, a humanized antibody only contains a humanized heavy chain. In specific embodiments, a humanized antibody only contains a humanized variable domain of a light chain and/or humanized heavy chain.

A humanized antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype including without limitation IgG1, IgG2, IgG3, and IgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions using techniques well known in the art.

The framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor antibody CDR or the consensus framework may be mutagenized by substitution, insertion and/or deletion of at least one amino acid residue so that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. In an exemplary embodiment, such mutations, however, will not be extensive. Usually, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. As used herein, the term “consensus framework” refers to the framework region in the consensus immunoglobulin sequence. As used herein, the term “consensus immunoglobulin sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.

With respect to constructing DVD-Ig or other binding protein molecules, a “linker” is used to denote a single amino acid or a polypeptide (“linker polypeptide”) comprising two or more amino acid residues joined by peptide bonds and used to link one or more antigen binding portions. Such linker polypeptides are well known in the art (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993); Poljak, R. J., Structure, 2: 1121-1123 (1994)). Exemplary linkers include, but are not limited to, SEQ ID NOs: 42 and 43; GGGGSG (SEQ ID NO:79), GGSGG (SEQ ID NO:80), GGGGSGGGGS (SEQ ID NO:81), GGSGGGGSG (SEQ ID NO:82), GGSGGGGSGS (SEQ ID NO:83), GGSGGGGSGGGGS (SEQ ID NO:84), GGGGSGGGGSGGGG (SEQ ID NO:85), GGGGSGGGGSGGGGS (SEQ ID NO:86), ASTKGPSVFPLAP (SEQ ID NO:87), RTVAAP (SEQ ID NO:88), TVAAPSVFIFPP (SEQ ID NO:89), RTVAAPSVFIFPP (SEQ ID NO:90), AKTTPKLEEGEFSEAR (SEQ ID NO:91), AKTTPKLEEGEFSEARV (SEQ ID NO:92), AKTTPKLGG (SEQ ID NO:93), SAKTTPKLGG (SEQ ID NO:94), SAKTTP (SEQ ID NO:95), RADAAP (SEQ ID NO:96), RADAAPTVS (SEQ ID NO:97), RADAAAAGGPGS (SEQ ID NO:98), RADAAAAGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:99), SAKTTPKLEEGEFSEARV (SEQ ID NO:100), ADAAP (SEQ ID NO:101), ADAAPTVSIFPP (SEQ ID NO:102), QPKAAP (SEQ ID NO:103), QPKAAPSVTLFPP (SEQ ID NO:104), AKTTPP (SEQ ID NO:105), AKTTPPSVTPLAP (SEQ ID NO:106), AKTTAP (SEQ ID NO:107), AKTTAPSVYPLAP (SEQ ID NO:108), GENKVEYAPALMALS (SEQ ID NO:109), GPAKELTPLKEAKVS (SEQ ID NO:110), and GHEAAAVMQVQYPAS (SEQ ID NO:111).

As used herein, “Vernier” zone refers to a subset of framework residues that may adjust CDR structure and fine-tune the fit to antigen as described by Foote and Winter, J. Mol. Biol., 224:487-499 (1992), which is incorporated herein by reference). Vernier zone residues form a layer underlying the CDRs and may impact on the structure of CDRs and the affinity of the antibody.

As used herein, the term “neutralizing” refers to neutralization of the biological activity of an antigen (e.g., the cytokines hIL-1α or IL-1β) when a binding protein specifically binds the antigen. Preferably, a neutralizing binding protein described herein binds to hIL-1α or hIL-1β resulting in the inhibition of a biological activity of hIL-1α or hIL-1β. Preferably, the neutralizing binding protein binds hIL-1β and reduces a biologically activity of hIL-1α or hIL-1β by at least about 20%, 40%, 60%, 80%, 85%, or more. Inhibition of a biological activity of hIL-1α or hIL-1β by a neutralizing binding protein can be assessed by measuring one or more indicators of hIL-1α or hIL-1β biological activity well known in the art. For example inhibition inhibition of human IL-8 secretion MRC-5 cells.

The term “activity” includes activities such as the binding specificity/affinity of an binding protein for an antigen, for example, an binding protein that specifically binds to an IL-1α antigen and IL-1β antigen and/or the neutralizing potency of an binding protein, for example, an anti-IL-1α/β binding protein whose binding to h IL-1α and β inhibits the biological activity of h IL-1α and β, for example, inhibition of human IL-8 secretion MRC-5 cells.

The term “epitope” includes any polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by a binding protein. In certain embodiments, a binding protein is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. Binding proteins are said to “bind to the same epitope” if the binding proteins cross-compete (one prevents the binding or modulating effect of the other). In addition, structural definitions of epitopes (overlapping, similar, identical) are informative, but functional definitions are often more relevant as they encompass structural (binding) and functional (modulation, competition) parameters.

The term “surface plasmon resonance”, as used herein, refers to an optical phenomenon that allows for the analysis of real-time b10 specific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further descriptions, see Jonsson et al., Ann. Biol. Clin., 51: 19-26 (1993); Jonsson et al., BioTechniques, 11: 620-627 (1991); Johnsson et al., J. Mol. Recognit., 8: 125-131 (1995); and Johnsson et al., Anal. Biochem., 198: 268-277 (1991).

The term “K_(on)” (also “Kon”, “kon”), as used herein, is intended to refer to the on rate constant for association of a binding protein (e.g., a DVD-Ig) to an antigen to form an association complex, e.g., binding protein/antigen complex, as is known in the art. The “K_(on)” also is known by the terms “association rate constant”, or “ka”, as used interchangeably herein. This value indicates the binding rate of a binding protein to its target antigen or the rate of complex formation between an antibody and antigen as is shown by the equation below:

Binding protein(“Ab”)+Antigen(“Ag”)→Ab-Ag.

The term “K_(off)” (also “Koff”, “koff”), as used herein, is intended to refer to the off rate constant for dissociation, or “dissociation rate constant”, of a binding protein (e.g., an DVD-Ig) from an association complex (e.g., a binding protein/antigen complex) as is known in the art. This value indicates the dissociation rate of an antibody from its target antigen or separation of Ab-Ag complex over time into free binding protein and antigen as shown by the equation below:

Ab+Ag←Ab-Ag.

The term “K_(D)” (also “K_(d)”), as used herein, is intended to refer to the “equilibrium dissociation constant”, and refers to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (Koff) by the association rate constant (Kon). The association rate constant (Kon), the dissociation rate constant (Koff), and the equilibrium dissociation constant (K are used to represent the binding affinity of a binding protein to an antigen. Methods for determining association and dissociation rate constants are well known in the art. Using fluorescence-based techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental approaches and instruments such as a BIAcore® (biomolecular interaction analysis) assay can be used (e.g., instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). Additionally, a KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Id.) can also be used.

The terms “label” and “detectable label” mean a moiety attached to a specific binding partner, such as a binding protein or an analyte, e.g., to render the reaction between members of a specific binding pair, such as a binding protein and an analyte, detectable. The specific binding partner, e.g., binding protein or analyte, so labeled is referred to as “detectably labeled”. Thus, the term “labeled binding protein” as used herein, refers to a protein with a label incorporated that provides for the identification of the binding protein. In an embodiment, the label is a detectable marker that can produce a signal that is detectable by visual or instrumental means, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin or streptavidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., ³H ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm), chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), and magnetic agents (e.g., gadolinium chelates). Representative examples of labels commonly employed for immunoassays include moieties that produce light, e.g., acridinium compounds, and moieties that produce fluorescence, e.g., fluorescein. Other labels are described herein. In this regard, the moiety itself may not be detectably labeled but may become detectable upon reaction with yet another moiety. Use of the term “detectably labeled” is intended to encompass the latter type of detectable labeling.

The term “binding protein conjugate” refers to a binding protein described herein chemically linked to a second chemical moiety, such as a therapeutic or cytotoxic agent. The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials. Preferably the therapeutic or cytotoxic agents include, but are not limited to, pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. When employed in the context of an immunoassay, a binding protein conjugate may be a detectably labeled antibody, which is used as the detection antibody.

The terms “crystal” and “crystallized” as used herein, refer to a binding protein (e.g., a DVD-Ig), or antigen binding portion thereof, that exists in the form of a crystal. Crystals are one form of the solid state of matter that is distinct from other forms such as the amorphous solid state or the liquid crystalline state. Crystals are composed of regular, repeating, three-dimensional arrays of atoms, ions, molecules (e.g., proteins such as DVD-Igs), or molecular assemblies (e.g., antigen/binding protein complexes). These three-dimensional arrays are arranged according to specific mathematical relationships that are well-understood in the field. The fundamental unit, or building block, that is repeated in a crystal is called the asymmetric unit. Repetition of the asymmetric unit in an arrangement that conforms to a given, well-defined crystallographic symmetry provides the “unit cell” of the crystal. Repetition of the unit cell by regular translations in all three dimensions provides the crystal. See Giege et al., Chapter 1, In Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ed., (Ducruix and Giege, eds.) (Oxford University Press, New York, 1999) pp. 1-16.

The term “polynucleotide” means a polymeric form of two or more nucleotides, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

The term “isolated polynucleotide” shall mean a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or some combination thereof) that, by virtue of its origin, the “isolated polynucleotide” is not associated with all or a portion of a polynucleotide with which the “isolated polynucleotide” is found in nature; is operably linked to a polynucleotide that it is not linked to in nature; or does not occur in nature as part of a larger sequence.

The term “vector”, as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. “Operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term “expression control sequence” as used herein refers to polynucleotide sequences that are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term “control sequences” is intended to include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

“Transformation”, as defined herein, refers to any process by which exogenous DNA enters a host cell. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. Such “transformed” cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. They also include cells which transiently express the inserted DNA or RNA for limited periods of time.

The term “recombinant host cell” (or simply “host cell”), is intended to refer to a cell into which exogenous DNA has been introduced. In an embodiment, the host cell comprises two or more (e.g., multiple) nucleic acids encoding antibodies, such as the host cells described in U.S. Pat. No. 7,262,028, for example. Such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. In an embodiment, host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life. In another embodiment, eukaryotic cells include protist, fungal, plant and animal cells. In another embodiment, host cells include but are not limited to the prokaryotic cell line Escherichia coli; mammalian cell lines CHO, HEK 293, COS, NSO, SP2 and PER.C6; the insect cell line Sf9; and the fungal cell Saccharomyces cerevisiae.

Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

“Transgenic organism”, as known in the art, refers to an organism having cells that contain a transgene, wherein the transgene introduced into the organism (or an ancestor of the organism) expresses a polypeptide not naturally expressed in the organism. A “transgene” is a DNA construct, which is stably and operably integrated into the genome of a cell from which a transgenic organism develops, directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic organism.

The terms “regulate” and “modulate” are used interchangeably, and, as used herein, refers to a change or an alteration in the activity of a molecule of interest (e.g., the biological activity of h IL-1α and β). Modulation may be an increase or a decrease in the magnitude of a certain activity or function of the molecule of interest. Exemplary activities and functions of a molecule include, but are not limited to, binding characteristics, enzymatic activity, cell receptor activation, and signal transduction.

Correspondingly, the term “modulator,” as used herein, is a compound capable of changing or altering an activity or function of a molecule of interest (e.g., the biological activity of hIL-1α and β). For example, a modulator may cause an increase or decrease in the magnitude of a certain activity or function of a molecule compared to the magnitude of the activity or function observed in the absence of the modulator. In certain embodiments, a modulator is an inhibitor, which decreases the magnitude of at least one activity or function of a molecule. Exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies, peptibodies, carbohydrates or small organic molecules. Peptibodies are described, e.g., in PCT Publication No. WO 01/83525.

The term “agonist”, as used herein, refers to a modulator that, when contacted with a molecule of interest, causes an increase in the magnitude of a certain activity or function of the molecule compared to the magnitude of the activity or function observed in the absence of the agonist. Particular agonists of interest may include, but are not limited to, IL-1α and/or β polypeptides, nucleic acids, carbohydrates, or any other molecule that binds to hIL-1α or β.

The terms “antagonist” and “inhibitor”, as used herein, refer to a modulator that, when contacted with a molecule of interest causes a decrease in the magnitude of a certain activity or function of the molecule compared to the magnitude of the activity or function observed in the absence of the antagonist. Particular antagonists of interest include those that block or modulate the biological or immunological activity of human IL-1α or β. Antagonists and inhibitors of human IL-1α or β may include, but are not limited to, proteins, nucleic acids, carbohydrates, or any other molecules, which bind to human IL-1α or β.

As used herein, the term “effective amount” refers to the amount of a therapy that is sufficient to reduce or ameliorate the severity and/or duration of a disorder or one or more symptoms thereof; prevent the advancement of a disorder; cause regression of a disorder; prevent the recurrence, development, onset, or progression of one or more symptoms associated with a disorder; detect a disorder; or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., prophylactic or therapeutic agent).

“Patient” and “subject” may be used interchangeably herein to refer to an animal, such as a mammal, including a primate (for example, a human, a monkey, and a chimpanzee), a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse or a whale), a bird (e.g., a duck or a goose), and a fish (e.g. zebrafish or a shark). Preferably, a patient or subject is a human, such as a human being treated or assessed for a disease, disorder or condition, a human at risk for a disease, disorder or condition, a human having a disease, disorder or condition, and/or human being treated for a disease, disorder or condition.

The term “sample”, as used herein, is used in its broadest sense. A “biological sample”, as used herein, includes, but is not limited to, any quantity of a substance from a living thing or formerly living thing. Such living things include, but are not limited to, humans, non-human primates, mice, rats, monkeys, dogs, rabbits and other animals. Such substances include, but are not limited to, blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes and spleen.

“Component”, “components,” and “at least one component,” refer generally to a capture antibody, a detection or conjugate antibody, a control, a calibrator, a series of calibrators, a sensitivity panel, a container, a buffer, a diluent, a salt, an enzyme, a co-factor for an enzyme, a detection reagent, a pretreatment reagent/solution, a substrate (e.g., as a solution), a stop solution, and the like that can be included in a kit for assay of a test sample, such as a patient urine, serum or plasma sample, in accordance with the methods described herein and other methods known in the art. Thus, in the context of the present disclosure, “at least one component,” “component,” and “components” can include a polypeptide or other analyte as above, such as a composition comprising an analyte such as polypeptide, which is optionally immobilized on a solid support, such as by binding to an anti-analyte (e.g., anti-polypeptide) binding protein. Some components can be in solution or lyophilized for reconstitution for use in an assay.

“Control” refers to a composition known to not analyte (“negative control”) or to contain analyte (“positive control”). A positive control can comprise a known concentration of analyte. “Control,” “positive control,” and “calibrator” may be used interchangeably herein to refer to a composition comprising a known concentration of analyte. A “positive control” can be used to establish assay performance characteristics and is a useful indicator of the integrity of reagents (e.g., analytes).

“Predetermined cutoff” and “predetermined level” refer generally to an assay cutoff value that is used to assess diagnostic/prognostic/therapeutic efficacy results by comparing the assay results against the predetermined cutoff/level, where the predetermined cutoff/level already has been linked or associated with various clinical parameters (e.g., severity of disease, progression/nonprogression/improvement, etc.). While the present disclosure may provide exemplary predetermined levels, it is well-known that cutoff values may vary depending on the nature of the immunoassay (e.g., antibodies employed, etc.). It further is well within the ordinary skill of one in the art to adapt the disclosure herein for other immunoassays to obtain immunoassay-specific cutoff values for those other immunoassays based on this disclosure. Whereas the precise value of the predetermined cutoff/level may vary between assays, correlations as described herein (if any) should be generally applicable.

“Pretreatment reagent,” e.g., lysis, precipitation and/or solubilization reagent, as used in a diagnostic assay as described herein is one that lyses any cells and/or solubilizes any analyte that is/are present in a test sample. Pretreatment is not necessary for all samples, as described further herein. Among other things, solubilizing the analyte (e.g., polypeptide of interest) may entail release of the analyte from any endogenous binding proteins present in the sample. A pretreatment reagent may be homogeneous (not requiring a separation step) or heterogeneous (requiring a separation step). With use of a heterogeneous pretreatment reagent there is removal of any precipitated analyte binding proteins from the test sample prior to proceeding to the next step of the assay.

“Quality control reagents” in the context of immunoassays and kits described herein, include, but are not limited to, calibrators, controls, and sensitivity panels. A “calibrator” or “standard” typically is used (e.g., one or more, such as a plurality) in order to establish calibration (standard) curves for interpolation of the concentration of an analyte, such as an antibody or an analyte. Alternatively, a single calibrator, which is near a predetermined positive/negative cutoff, can be used. Multiple calibrators (i.e., more than one calibrator or a varying amount of calibrator(s)) can be used in conjunction so as to comprise a “sensitivity panel.”

“Risk” refers to the possibility or probability of a particular event occurring either presently or at some point in the future. “Risk stratification” refers to an array of known clinical risk factors that allows physicians to classify patients into a low, moderate, high or highest risk of developing a particular disease, disorder or condition.

“Specific” and “specificity” in the context of an interaction between members of a specific binding pair (e.g., an antigen (or fragment thereof) and a binding protein (or antigenically reactive fragment thereof)) refer to the selective reactivity of the interaction. The phrase “specifically binds to” and analogous phrases refer to the ability of binding proteins to bind specifically to analyte (or a fragment thereof) and not bind specifically to other entities.

“Specific binding partner” is a member of a specific binding pair. A specific binding pair comprises two different molecules, which specifically bind to each other through chemical or physical means. Therefore, in addition to antigen and binding protein specific binding pairs of common immunoassays, other specific binding pairs can include biotin and avidin (or streptavidin), carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding members, for example, an analyte-analog. Immunoreactive specific binding members include antigens, antigen fragments, and antibodies, including monoclonal and polyclonal antibodies as well as complexes, fragments, and variants (including fragments of variants) thereof, whether isolated or recombinantly produced.

“Variant” as used herein means a polypeptide that differs from a given polypeptide (e.g., binding proteins or IL-1α or (3 polypeptide) in amino acid sequence by the addition (e.g., insertion), deletion, or conservative substitution of amino acids, but that retains the biological activity of the given polypeptide (e.g., a variant IL-1β can compete with anti-IL-1β binding protein for binding to IL-1β.) A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity and degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art (see, e.g., Kyte et al., J. Mol. Biol., 157: 105-132 (1982)). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids also can be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity (see, e.g., U.S. Pat. No. 4,554,101). Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. In one aspect, substitutions are performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties. “Variant” also can be used to describe a polypeptide or fragment thereof that has been differentially processed, such as by proteolysis, phosphorylation, or other post-translational modification, yet retains its biological activity or antigen reactivity, e.g., the ability to bind to IL-1β. Use of “variant” herein is intended to encompass fragments of a variant unless otherwise contradicted by context.

Alternatively or additionally, a “variant” is to be understood as a polynucleotide or protein which differs in comparison to the polynucleotide or protein from which it is derived by one or more changes in its length or sequence. The polypeptide or polynucleotide from which a protein or nucleic acid variant is derived is also known as the parent polypeptide or polynucleotide. The term “variant” comprises “fragments” or “derivatives” of the parent molecule. Typically, “fragments” are smaller in length or size than the parent molecule, whilst “derivatives” exhibit one or more differences in their sequence in comparison to the parent molecule. Also encompassed modified molecules such as but not limited to post-translationally modified proteins (e.g. glycosylated, biotinylated, phosphorylated, ubiquitinated, palmitoylated, or proteolytically cleaved proteins) and modified nucleic acids such as methylated DNA. Also mixtures of different molecules such as but not limited to RNA-DNA hybrids, are encompassed by the term “variant”. Typically, a variant is constructed artificially, preferably by gene-technological means whilst the parent polypeptide or polynucleotide is a wild-type protein or polynucleotide. However, also naturally occurring variants are to be understood to be encompassed by the term “variant” as used herein. Further, the variants usable in the present disclosure may also be derived from homologs, orthologs, or paralogs of the parent molecule or from artificially constructed variant, provided that the variant exhibits at least one biological activity of the parent molecule, i.e. is functionally active.

Alternatively or additionally, a “variant” as used herein, can be characterized by a certain degree of sequence identity to the parent polypeptide or parent polynucleotide from which it is derived. More precisely, a protein variant in the context of the present disclosure exhibits at least 80% sequence identity to its parent polypeptide. A polynucleotide variant in the context of the present disclosure exhibits at least 80% sequence identity to its parent polynucleotide. The term “at least 80% sequence identity” is used throughout the specification with regard to polypeptide and polynucleotide sequence comparisons. This expression preferably refers to a sequence identity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the respective reference polypeptide or to the respective reference polynucleotide.

The similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity, can be determined via sequence alignments. Such alignments can be carried out with several art-known algorithms, preferably with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) or with the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80) available e.g. on http://www.ebi.ac.uk/Tools/clustalw/ or on http://www.ebi.ac.uk/Tools/clustalw2/index.html or on http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html. Preferred parameters used are the default parameters as they are set on http://www.ebi.ac.uk/Tools/clustalw/ or http://www.ebi.ac.uk/Tools/clustalw2/index.html. The grade of sequence identity (sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215: 403-410. BLAST polynucleotide searches are performed with the BLASTN program, score=100, word length=12, to obtain polynucleotide sequences that are homologous to those nucleic acids which encode mir-146a. BLAST protein searches are performed with the BLASTP program, score=50, word length=3, to obtain amino acid sequences homologous to mir-146a. To obtain gapped alignments for comparative purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:I54-I62) or Markov random fields. When percentages of sequence identity are referred to in the present application, these percentages are calculated in relation to the full length of the longer sequence, if not specifically indicated otherwise.

I. Method of Making Anti-IL-1α or β Antibodies

Anti-IL-1α or β antibodies of the present disclosure may be made by any of a number of techniques known in the art. In particular, methods of making anti-IL-1β antibodies are described in U.S. Patent Publication No. 2011/0280800, incorporated by reference, herein, in its entirety.

B. Anti IL-1α/β DVD-Ig™ Binding Proteins

Provided herein are dual variable domain immunoglobulin binding proteins (DVD-Igs) that bind one or more epitopes of IL-1α and/or β. A DVD-Ig binding protein may also bind an epitope of IL-1α or β and an epitope of a second target antigen other than an IL-1α or β polypeptide. An exemplary embodiment of such DVD-Ig molecules comprises a heavy chain that comprises the structural formula VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker with the proviso that it is not CH1, X2 is an Fc region, and n is 0 or 1, and preferably 1; and a light chain that comprises the structural formula VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker with the proviso that it is not CH1, and X2 does not comprise an Fc region; and n is 0 or 1, and preferably 1. Such a DVD-Ig may comprise two such heavy chains and two such light chains, wherein each chain comprises variable domains linked in tandem without an intervening constant region between variable regions, wherein a heavy chain and a light chain associate to form two tandem antigen binding sites, and a pair of heavy and light chains may associate with another pair of heavy and light chains to form a tetrameric binding protein with four antigen binding sites. In another embodiment, a DVD-Ig molecule may comprise heavy and light chains that each comprise three variable domains, e.g., VD1, VD2, VD3, linked in tandem without an intervening constant region between variable domains, wherein a pair of heavy and light chains may associate to form three antigen binding sites, and wherein a pair of heavy and light chains may associate with another pair of heavy and light chains to form a tetrameric binding protein with six antigen binding sites.

Each variable domain (VD) in a DVD-Ig may be obtained from one or more “parent” monoclonal antibodies that bind one or more desired antigens or epitopes, such as IL-1β and/or IL-1α antigens or epitopes. General methods of making DVD-Ig and properties associated with DVD-Igs are described in U.S. Patent Publication No. 2011/0280800, incorporated by reference, herein, in its entirety. Specific methods used with the DVD-Ig specifically presented herein are provided below.

III. Use of DVD-Igs in Various Diseases

DVD-Ig molecules of the disclosure are also useful as therapeutic molecules to treat various diseases. Such DVD molecules may bind one or more targets involved in a specific disease. Examples of such targets in various diseases are described below.

IL-1 family members (IL-1β and IL-1α) play a critical role in the pathology associated with a variety of disorders involving immune and inflammatory elements. An IL-1 binding protein described herein may be administered to an individual to treat such disorders. In an embodiment, a disorder that may be treated by a method of the disclosure comprising administering to a subject an IL-1 binding protein described herein includes, but is not limited to, diabetes; uveitis; neuropathic pain; osteoarthritic pain; inflammatory pain; rheumatoid arthritis; osteoarthritis; juvenile chronic arthritis; septic arthritis; Lyme arthritis; psoriatic arthritis; reactive arthritis; spondyloarthropathy; systemic lupus erythematosus (SLE); Crohn's disease; ulcerative colitis; inflammatory bowel disease; autoimmune diabetes; insulin dependent diabetes mellitus; thyroiditis; asthma; allergic diseases; psoriasis; dermatitis; scleroderma; graft versus host disease; organ transplant rejection; acute immune disease associated with organ transplantation; chronic immune disease associated with organ transplantation; sarcoidosis; atherosclerosis; disseminated intravascular coagulation (DIC); Kawasaki's disease; Grave's disease; nephrotic syndrome; chronic fatigue syndrome; Wegener's granulomatosis; Henoch-Schoenlein purpurea; microscopic vasculitis of the kidneys; chronic active hepatitis; autoimmune uveitis; septic shock; toxic shock syndrome; sepsis syndrome; cachexia; infectious diseases; parasitic diseases; acute transverse myelitis; Huntington's chorea; Parkinson's disease; Alzheimer's disease; stroke; primary biliary cirrhosis; hemolytic anemia; malignancies; heart failure; myocardial infarction; Addison's disease; sporadic polyglandular deficiency type I; polyglandular deficiency type II (Schmidt's syndrome); acute respiratory distress syndrome (ARDS); alopecia; alopecia greata; seronegative arthropathy; arthropathy; Reiter's disease; psoriatic arthropathy; ulcerative colitic arthropathy; enteropathic synovitis; chlamydia; Yersinia and Salmonella associated arthropathy; spondyloarthropathy; atheromatous disease/arteriosclerosis; atopic allergy; autoimmune bullous disease; pemphigus vulgaris; pemphigus foliaceus; pemphigoid; linear IgA disease; autoimmune haemolytic anemia; Coombs positive haemolytic anemia; acquired pernicious anemia; juvenile pernicious anemia; myalgic encephalitis/Royal Free disease; chronic mucocutaneous candidiasis; giant cell arteritis (GCA); primary sclerosing hepatitis; cryptogenic autoimmune hepatitis; acquired immunodeficiency syndrome (AIDS); acquired immunodeficiency related diseases; hepatitis B; hepatitis C; common varied immunodeficiency (common variable hypogammaglobulinaemia); dilated cardiomyopathy; female infertility; ovarian failure; premature ovarian failure; fibrotic lung disease; cryptogenic fibrosing alveolitis; post-inflammatory interstitial lung disease; interstitial pneumonitis; connective tissue disease associated interstitial lung disease; mixed connective tissue disease associated lung disease; systemic sclerosis associated interstitial lung disease; rheumatoid arthritis associated interstitial lung disease; systemic lupus erythematosus associated lung disease; dermatomyositis/polymyositis associated lung disease; Sjorgren's disease associated lung disease; ankylosing spondylitis associated lung disease; vasculitic diffuse lung disease; haemosiderosis associated lung disease; drug-induced interstitial lung disease; fibrosis; radiation fibrosis; bronchiolitis obliterans; chronic eosinophilic pneumonia; lymphocytic infiltrative lung disease; postinfectious interstitial lung disease; gouty arthritis; autoimmune hepatitis; type-1 autoimmune hepatitis (classical autoimmune or lupoid hepatitis); type-2 autoimmune hepatitis (anti-LKM antibody hepatitis); autoimmune mediated hypoglycemia; type B insulin resistance with acanthosis nigricans; hypoparathyroidism; osteoarthritis; primary sclerosing cholangitis; psoriasis type 1; psoriasis type 2; idiopathic leucopenia; autoimmune neutropaenia; renal disease NOS; glomerulonephritides; microscopic vasculitis of the kidneys; Lyme disease; discoid lupus erythematosus; idiopathic male infertility; nitric oxide-associated male infertility; sperm autoimmunity; multiple sclerosis (all subtypes, including primary progressive, secondary progressive, relapsing remitting); sympathetic ophthalmia; pulmonary hypertension secondary to connective tissue disease; Goodpasture's syndrome; pulmonary manifestation of polyarteritis nodosa; acute rheumatic fever; rheumatoid spondylitis; Still's disease; systemic sclerosis; Sjorgren's syndrome; Takayasu's disease/arteritis; autoimmune thrombocytopenia (AITP); idiopathic thrombocytopenia; autoimmune thyroid disease; hyperthyroidism; goitrous autoimmune hypothyroidism (Hashimoto's disease); atrophic autoimmune hypothyroidism; primary myxoedema; phacogenic uveitis; primary vasculitis; vitiligo; acute liver disease; chronic liver disease; alcoholic cirrhosis; alcohol-induced liver injury; cholestasis; hypercholesterolemia; idiosyncratic liver disease; drug-induced hepatitis; non-alcoholic steatohepatitis; allergy; group B Streptococci (GBS) infection; mental disorders (e.g., depression and schizophrenia); Th2 Type and Th1 Type mediated diseases; acute and chronic pain (different forms of pain); cancer (such as lung, breast, stomach, bladder, colon, pancreas, ovarian, prostate, and rectal cancer); hematopoietic malignancies; leukemia; lymphoma; abetalipoproteinemia; acrocyanosis; acute and chronic parasitic or infectious processes; acute leukemia; acute lymphoblastic leukemia (ALL); T-cell ALL; FAB ALL; acute myeloid leukemia (AML); acute or chronic bacterial infection; acute pancreatitis; acute renal failure; adenocarcinomas; atrial ectopic beats; AIDS dementia complex; alcohol-induced hepatitis; allergic conjunctivitis; allergic contact dermatitis; allergic rhinitis; allograft rejection; alpha-1-antitrypsin deficiency; amyotrophic lateral sclerosis; anemia; angina pectoris; anterior horn cell degeneration; anti-CD3 therapy; antiphospholipid syndrome; anti-receptor hypersensitivity reactions; aortic and peripheral aneurysms; aortic dissection; arterial hypertension; arteriosclerosis; arteriovenous fistula; ataxia; atrial fibrillation (sustained or paroxysmal); atrial flutter; atrioventricular block; B cell lymphoma; bone graft rejection; bone marrow transplant (BMT) rejection; bundle branch block; Burkitt's lymphoma; burns; cardiac arrhythmias; cardiac stun syndrome; cardiac tumors; cardiomyopathy; cardiopulmonary bypass inflammation response; cartilage transplant rejection; cerebellar cortical degenerations; cerebellar disorders; chaotic or multifocal atrial tachycardia; chemotherapy associated disorders; chronic myelocytic leukemia (CML); chronic alcoholism; chronic inflammatory pathologies; chronic lymphocytic leukemia (CLL); chronic obstructive pulmonary disease (COPD); chronic salicylate intoxication; colorectal carcinoma; congestive heart failure; conjunctivitis; contact dermatitis; cor pulmonale; coronary artery disease; Creutzfeldt-Jakob disease; culture negative sepsis; cystic fibrosis; cytokine therapy associated disorders; dementia pugilistica; demyelinating diseases; dengue hemorrhagic fever; dermatitis; dermatologic conditions; diabetes mellitus; diabetic arteriosclerotic disease; diffuse Lewy body disease; dilated congestive cardiomyopathy; disorders of the basal ganglia; Down's syndrome in middle age; drug-induced movement disorders induced by drugs which block CNS dopamine receptors; drug sensitivity; eczema; encephalomyelitis; endocarditis; endocrinopathy; epiglottitis; Epstein-Barr virus infection; erythromelalgia; extrapyramidal and cerebellar disorders; familial hemophagocytic lymphohistiocytosis; fetal thymus implant rejection; Friedreich's ataxia; functional peripheral arterial disorders; fungal sepsis; gas gangrene; gastric ulcer; glomerular nephritis; graft rejection of any organ or tissue; gram negative sepsis; gram positive sepsis; granulomas due to intracellular organisms; hairy cell leukemia; Hallervorden-Spatz disease; Hashimoto's thyroiditis; hay fever; heart transplant rejection; hemochromatosis; hemodialysis; hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura; hemorrhage; hepatitis A; His bundle arrhythmias; HIV infection/HIV neuropathy; Hodgkin's disease; hyperkinetic movement disorders; hypersensitivity reactions; hypersensitivity pneumonitis; hypertension; hypokinetic movement disorders; hypothalamic-pituitary-adrenal axis evaluation; idiopathic Addison's disease; idiopathic pulmonary fibrosis (IPF); antibody mediated cytotoxicity; asthenia; infantile spinal muscular atrophy; inflammation of the aorta; influenza a; ionizing radiation exposure; iridocyclitis/uveitis/optic neuritis; ischemia-reperfusion injury; ischemic stroke; juvenile rheumatoid arthritis; juvenile spinal muscular atrophy; Kaposi's sarcoma; kidney transplant rejection; legionella; leishmaniasis; leprosy; lesions of the corticospinal system; lipedema; liver transplant rejection; lymphedema; malaria; malignant lymphoma; malignant histiocytosis; malignant melanoma; meningitis; meningococcemia; metabolic syndrome migraine headache; idiopathic migraine headache; mitochondrial multisystem disorder; mixed connective tissue disease; monoclonal gammopathy; multiple myeloma; multiple systems degenerations (Menzel; Dejerine-Thomas; Shy-Drager; and Machado-Joseph); myasthenia gravis; mycobacterium avium intracellulare; mycobacterium tuberculosis; myelodysplastic syndrome; myocardial infarction; myocardial ischemic disorders; nasopharyngeal carcinoma; neonatal chronic lung disease; nephritis; nephrosis; neurodegenerative diseases; neurogenic I muscular atrophies; neutropenic fever; non-Hodgkin's lymphoma; occlusion of the abdominal aorta and its branches; occlusive arterial disorders; OKT3® therapy; orchitis/epididymitis; orchitis/vasectomy reversal procedures; organomegaly; osteoporosis; pancreas transplant rejection; pancreatic carcinoma; paraneoplastic syndrome/hypercalcemia of malignancy; parathyroid transplant rejection; pelvic inflammatory disease; perennial rhinitis; pericardial disease; peripheral atherosclerotic disease; peripheral vascular disorders; peritonitis; pernicious anemia; pneumocystis carinii pneumonia; pneumonia; POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes syndrome); post perfusion syndrome; post pump syndrome; post-MI cardiotomy syndrome; preeclampsia; progressive supra nucleo palsy; primary pulmonary hypertension; radiation therapy; Raynaud's phenomenon; Raynaud's disease; Refsum's disease; regular narrow QRS tachycardia; renovascular hypertension; reperfusion injury; restrictive cardiomyopathy; sarcomas; senile chorea; senile dementia of Lewy body type; seronegative arthropathies; shock; sickle cell anemia; skin allograft rejection; skin changes syndrome; small bowel transplant rejection; solid tumors; specific arrhythmias; spinal ataxia; spinocerebellar degenerations; streptococcal myositis; structural lesions of the cerebellum; subacute sclerosing panencephalitis; syncope; syphilis of the cardiovascular system; systemic anaphylaxis; systemic inflammatory response syndrome; systemic onset juvenile rheumatoid arthritis; telangiectasia; thromboangitis obliterans; thrombocytopenia; toxicity; transplants; trauma/hemorrhage; type III hypersensitivity reactions; type IV hypersensitivity; unstable angina; uremia; urosepsis; urticaria; valvular heart diseases; varicose veins; vasculitis; venous diseases; venous thrombosis; ventricular fibrillation; viral and fungal infections; viral encephalitis/aseptic meningitis; viral-associated hemophagocytic syndrome; Wernicke-Korsakoff syndrome; Wilson's disease; xenograft rejection of any organ or tissue; acute coronary syndromes; acute idiopathic polyneuritis; acute inflammatory demyelinating polyradiculoneuropathy; acute ischemia; adult Still's disease; alopecia greata; anaphylaxis; anti-phospholipid antibody syndrome; aplastic anemia; arteriosclerosis; atopic eczema; atopic dermatitis; autoimmune dermatitis; autoimmune disorder associated with Streptococcus infection; autoimmune enteropathy; autoimmune hearing loss; autoimmune lymphoproliferative syndrome (ALPS); autoimmune myocarditis; autoimmune premature ovarian failure; blepharitis; bronchiectasis; bullous pemphigoid; cardiovascular disease; catastrophic antiphospholipid syndrome; celiac disease; cervical spondylosis; chronic ischemia; cicatricial pemphigoid; clinically isolated syndrome (CIS) with risk for multiple sclerosis; conjunctivitis; childhood onset psychiatric disorder; dacryocystitis; dermatomyositis; diabetic retinopathy; disk herniation; disk prolapse; drug induced immune hemolytic anemia; endocarditis; endometriosis; endophthalmitis; episcleritis; erythema multiforme; erythema multiforme major; gestational pemphigoid; Guillain-Barre syndrome (GBS); hay fever; Hughes syndrome; idiopathic Parkinson's disease; idiopathic interstitial pneumonia; IgE-mediated allergy; immune hemolytic anemia; inclusion body myositis; infectious ocular inflammatory disease; inflammatory demyelinating disease; inflammatory heart disease; inflammatory kidney disease; iritis; keratitis; keratojunctivitis sicca; Kussmaul disease or Kussmaul-Meier disease; Landry's paralysis; Langerhan's cell histiocytosis; livedo reticularis; macular degeneration; microscopic polyangiitis; Morbus Bechterev; motor neuron disorders; mucous membrane pemphigoid; multiple organ failure; myasthenia gravis; myelodysplastic syndrome; myocarditis; nerve root disorders; neuropathy; non-A non-B hepatitis; optic neuritis; osteolysis; pauciarticular JRA; peripheral artery occlusive disease (PAOD); peripheral vascular disease (PVD); peripheral artery; disease (PAD); phlebitis; polyarteritis nodosa (or periarteritis nodosa); polychondritis; polymyalgia rheumatica; poliosis; polyarticular JRA; polyendocrine deficiency syndrome; polymyositis; polymyalgia rheumatica (PMR); post-pump syndrome; primary Parkinsonism; secondary Parkinsonism; prostatitis; pure red cell aplasia; primary adrenal insufficiency; recurrent neuromyelitis optica; restenosis; rheumatic heart disease; SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis); secondary amyloidosis; shock lung; scleritis; sciatica; secondary adrenal insufficiency; silicone associated connective tissue disease; Sneddon-Wilkinson dermatosis; spondylitis ankylosans; Stevens-Johnson syndrome (SJS); systemic inflammatory response syndrome; temporal arteritis; toxoplasmic retinitis; toxic epidermal necrolysis; transverse myelitis; TRAPS (tumor necrosis factor receptor type 1 (TNFR)-associated periodic syndrome); type B insulin resistance with acanthosis nigricans; type 1 allergic reaction; type II diabetes; urticaria; usual interstitial pneumonia (UIP); vernal conjunctivitis; viral retinitis; Vogt-Koyanagi-Harada syndrome (VKH syndrome); wet macular degeneration; wound healing; and Yersinia and Salmonella associated arthropathy.

The binding proteins of the disclosure can be used to treat humans suffering from autoimmune diseases, in particular those associated with inflammation, rheumatoid arthritis (RA), osteoarthritis, psoriasis, multiple sclerosis (MS), and other autoimmune diseases.

Allergic asthma is characterized by the presence of eosinophilia, goblet cell metaplasia, epithelial cell alterations, airway hyperreactivity (AHR), and Th2 and Th1 cytokine expression, as well as elevated serum IgE levels. It is now widely accepted that airway inflammation is the key factor underlying the pathogenesis of asthma, involving a complex interplay of inflammatory cells such as T cells, B cells, eosinophils, mast cells and macrophages, and of their secreted mediators including cytokines and chemokines. Corticosteroids are the most important anti-inflammatory treatment for asthma today, however their mechanism of action is non-specific and safety concerns exist, especially in the juvenile patient population. The development of more specific and targeted therapies is therefore warranted.

Animal models such as OVA-induced asthma mouse model, where both inflammation and AHR can be assessed, are known in the art and may be used to determine the ability of various DVD-Ig molecules to treat asthma. Animal models for studying asthma are disclosed in Coffman et al., J. Exp. Med., 201(12): 1875-1879 (2005); Lloyd et al., Adv. Immunol., 77: 263-295 (2001); Boyce et al., J. Exp. Med., 201(12): 1869-1873 (2005); and Snibson et al., Clin. Exp. Allergy, 35(2): 146-152 (2005). In addition to routine safety assessments of these target pairs specific tests for the degree of immunosuppression may be warranted and helpful in selecting the best target pairs (see Luster et al., Toxicology, 92(1-3): 229-243 (1994); Descotes, J., Develop. Biol. Standard., 77: 99-102 (1992); Hart et al., J. Allergy Clin. Immunol., 108(2): 250-257 (2001)).

One aspect of the disclosure includes a dual-specific anti-IL-1β/IL-1α DVD-Ig as a therapeutic agent beneficial for the treatment of asthma.

Osteoarthritis

The articular cartilage, or “hyaline cartilage”, of healthy vertebrates (including humans and other mammals) is a semi-transparent, opalescent connective tissue characterized by a columnar growth pattern of chondrocytes in an extracellular matrix (ECM) composed predominantly of proteoglycans, type II collagen, and water. Articular cartilage provides an effective weight-bearing cushion to prevent contact between opposing bones in a joint and thus is critical to the normal function of the joint. Articular cartilage is not only susceptible to damage by joint trauma, but also to a gradual process of erosion. Initially, such an erosion may be simply an asymptomatic “partial thickness defect” in which an area of reduced hyaline cartilage does not penetrate completely to the subchondral bone. Such partial thickness defects are usually not painful and typically are only detected during arthroscopic examination. However, if the erosive process is not treated, the base of a partial thickness defect may continue to wear away and the diameter of the defect may increase such that the defect eventually progresses to a “full thickness defect” that penetrates the underlying bone. Such full thickness defects may become sufficiently large that surfaces of opposing bones of the joint make contact and begin to erode one another, leading to inflammation, pain, and other degenerative changes, i.e., the classic symptoms of osteoarthritis (OA). Osteoarthritis is thus a degenerative, progressive, and crippling disease that results in joint deformity, instability, impairment, and pain. Eventually, joint replacement surgery may be the only practical recourse for restoring, at least in part, some level of mobility to an individual.

One aspect of the disclosure includes a dual-specific anti-IL-1β/IL-1α DVD-Ig as a therapeutic agent beneficial for the treatment of asthma.

Pain

The invention also provides a method of treating pain in an individual (human or other mammal) comprising the step of administering to the individual a protein that binds IL-1α and another protein that binds IL-1β. In an embodiment, the binding proteins are administered in combination, for example, in a mixture, by successive administration, or by concurrent administration. Binding proteins useful in a method of treating pain according to the invention include, but are not limited to, antibodies that bind IL-1α and antibodies that bind IL-1β.

In another aspect of the invention, a method of treating pain in an individual comprises the step of administering to the individual a dual variable domain immunoglobulin binding protein (also referred herein as “DVD-Ig™” or “DVD-Ig” binding protein or molecule) that comprises at least one antigen binding site that binds IL-1α and at least one antigen binding site that binds IL-1β.

In an embodiment, the invention provides a method of treating pain in an individual suffering from a disease or disorder associated with IL-1 accumulation. Such IL-1 accumulation in the individual can be the result of reduced IL-1 expression or reduced metabolism of IL-1. Accumulation of IL-1 may be in plasma or local tissue of the individual.

In an embodiment, the invention provides a method for treating an individual for pain wherein the individual is suffering from a disease or disorder associated with IL-1 accumulation.

In an embodiment, the compositions and methods described herein can be used to treat pain in an individual suffering from a disease or disorder selected from the group comprising osteoarthritis, rheumatoid arthritis, juvenile chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythemato sus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, insulin dependent diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, dermatitis scleroderma, graft versus host disease, organ transplant rejection, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignancies, heart failure, myocardial infarction, Addison's disease, sporadic polyglandular deficiency type I and polyglandular deficiency type II, Schmidt's syndrome, adult (acute) respiratory distress syndrome, alopecia, alopecia greata, seronegative arthopathy, arthropathy, Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy, enteropathic synovitis, chlamydia, yersinia and salmonella associated arthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis, atopic allergy, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune haemolytic anaemia, Coombs positive haemolytic anaemia, acquired pernicious anaemia, juvenile pernicious anaemia, myalgic encephalitis/Royal Free Disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome, Acquired Immunodeficiency Related Diseases, Hepatitis B, Hepatitis C, common varied immunodeficiency (common variable hypogammaglobulinaemia), dilated cardiomyopathy, female infertility, ovarian failure, premature ovarian failure, fibrotic lung disease, cryptogenic fibrosing alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonitis, connective tissue disease associated interstitial lung disease, mixed connective tissue disease associated lung disease, systemic sclerosis associated interstitial lung disease, rheumatoid arthritis associated interstitial lung disease, systemic lupus erythematosus associated lung disease, dermatomyositis/polymyositis associated lung disease, Sjögren's disease associated lung disease, ankylosing spondylitis associated lung disease, vasculitic diffuse lung disease, haemosiderosis associated lung disease, drug-induced interstitial lung disease, fibrosis, radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type-1 autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune mediated hypoglycemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune disease associated with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthrosis, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leucopaenia, autoimmune neutropaenia, renal disease NOS, glomerulonephritides, microscopic vasulitis of the kidneys, lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS, sperm autoimmunity, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestation of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjorgren's syndrome, Takayasu's disease/arteritis, autoimmune thrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute liver disease, chronic liver diseases, alcoholic cirrhosis, alcohol-induced liver injury, cholestasis, idiosyncratic liver disease, Drug-Induced hepatitis, Non-alcoholic Steatohepatitis, allergy and asthma, group B streptococci (GBS) infection, mental disorders (e.g., depression and schizophrenia), Th2 Type and Th1 Type mediated diseases, acute and chronic pain (different forms of pain), and cancers such as lung, breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectal cancer and hematopoietic malignancies (leukemia and lymphoma), Abetalipoprotemia, Acrocyanosis, acute and chronic parasitic or infectious processes, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute or chronic bacterial infection, acute pancreatitis, acute renal failure, adenocarcinomas, aerial ectopic beats, AIDS dementia complex, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allograft rejection, alpha-1-antitryp sin deficiency, amyotrophic lateral sclerosis, anemia, angina pectoris, anterior horn cell degeneration, anti cd3 therapy, antiphospholipid syndrome, anti-receptor hypersensitivity reactions, aortic and peripheral aneuryisms, aortic dissection, arterial hypertension, arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation (sustained or paroxysmal), atrial flutter, atrioventricular block, B cell lymphoma, bone graft rejection, bone marrow transplant (BMT) rejection, bundle branch block, Burkitt's lymphoma, Burns, cardiac arrhythmias, cardiac stun syndrome, cardiac tumors, cardiomyopathy, cardiopulmonary bypass inflammation response, cartilage transplant rejection, cerebellar cortical degenerations, cerebellar disorders, chaotic or multifocal atrial tachycardia, chemotherapy associated disorders, chronic myelocytic leukemia (CML), chronic alcoholism, chronic inflammatory pathologies, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylate intoxication, colorectal carcinoma, congestive heart failure, conjunctivitis, contact dermatitis, cor pulmonale, coronary artery disease, Creutzfeldt-Jakob disease, culture negative sepsis, cystic fibrosis, cytokine therapy associated disorders, Dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever, dermatitis, dermatologic conditions, diabetes, diabetes mellitus, diabetic ateriosclerotic disease, Diffuse Lewy body disease, dilated congestive cardiomyopathy, disorders of the basal ganglia, Down's Syndrome in middle age, drug-induced movement disorders induced by drugs which block CNS dopamine receptors, drug sensitivity, eczema, encephalomyelitis, endocarditis, endocrinopathy, epiglottitis, epstein-barr virus infection, erythromelalgia, extrapyramidal and cerebellar disorders, familial hematophagocytic lymphohistiocytosis, fetal thymus implant rejection, Friedreich's ataxia, functional peripheral arterial disorders, fungal sepsis, gas gangrene, gastric ulcer, glomerular nephritis, graft rejection of any organ or tissue, gram negative sepsis, gram positive sepsis, granulomas due to intracellular organisms, hairy cell leukemia, Hallerrorden-Spatz disease, hashimoto's thyroiditis, hay fever, heart transplant rejection, hemachromatosis, hemodialysis, hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura, hemorrhage, hepatitis (A), His bundle arrythmias, HIV infection/HIV neuropathy, Hodgkin's disease, hyperkinetic movement disorders, hypersensitity reactions, hypersensitivity pneumonitis, hypertension, hypokinetic movement disorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathic Addison's disease, idiopathic pulmonary fibrosis, antibody mediated cytotoxicity, Asthenia, infantile spinal muscular atrophy, inflammation of the aorta, influenza a, ionizing radiation exposure, iridocyclitis/uveitis/optic neuritis, ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma, kidney transplant rejection, legionella, leishmaniasis, leprosy, lesions of the corticospinal system, lipedema, liver transplant rejection, lymphederma, malaria, malignamt Lymphoma, malignant histiocytosis, malignant melanoma, meningitis, meningococcemia, metabolic/idiopathic diseases, migraine headache, mitochondrial multi.system disorder, mixed connective tissue disease, monoclonal gammopathy, multiple myeloma, multiple systems degenerations (Mencel Dejerine-Thomas Shi-Drager and Machado-Joseph), myasthenia gravis, mycobacterium avium intracellulare, mycobacterium tuberculosis, myelodyplastic syndrome, myocardial infarction, myocardial ischemic disorders, nasopharyngeal carcinoma, neonatal chronic lung disease, nephritis, nephrosis, neurodegenerative diseases, neurogenic I muscular atrophies, neutropenic fever, non-hodgkins lymphoma, occlusion of the abdominal aorta and its branches, occlusive arterial disorders, okt3 therapy, orchitis/epidydimitis, orchitis/vasectomy reversal procedures, organomegaly, osteoporosis, pancreas transplant rejection, pancreatic carcinoma, paraneoplastic syndrome/hypercalcemia of malignancy, parathyroid transplant rejection, pelvic inflammatory disease, perennial rhinitis, pericardial disease, peripheral atherlosclerotic disease, peripheral vascular disorders, peritonitis, pernicious anemia, pneumocystis carinii pneumonia, pneumonia, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes syndrome), post perfusion syndrome, post pump syndrome, post-MI cardiotomy syndrome, preeclampsia, Progressive supranucleo Palsy, primary pulmonary hypertension, radiation therapy, Raynaud's phenomenon and disease, Raynoud's disease, Refsum's disease, regular narrow QRS tachycardia, renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea, Senile Dementia of Lewy body type, seronegative arthropathies, shock, sickle cell anemia, skin allograft rejection, skin changes syndrome, small bowel transplant rejection, solid tumors, specific arrythmias, spinal ataxia, spinocerebellar degenerations, streptococcal myositis, structural lesions of the cerebellum, Subacute sclerosing panencephalitis, Syncope, syphilis of the cardiovascular system, systemic anaphalaxis, systemic inflammatory response syndrome, systemic onset juvenile rheumatoid arthritis, T-cell or FAB ALL, Telangiectasia, thromboangitis obliterans, thrombocytopenia, toxicity, transplants, trauma/hemorrhage, type III hypersensitivity reactions, type IV hypersensitivity, unstable angina, uremia, urosepsis, urticaria, valvular heart diseases, varicose veins, vasculitis, venous diseases, venous thrombosis, ventricular fibrillation, viral and fungal infections, vital encephalitis/aseptic meningitis, vital-associated hemaphagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's disease, xenograft rejection of any organ or tissue, acute coronary syndromes, acute idiopathic polyneuritis, acute inflammatory demyelinating polyradiculoneuropathy, acute ischemia, adult Still's disease, alopecia greata, anaphylaxis, anti-phospholipid antibody syndrome, aplastic anemia, arteriosclerosis, atopic eczema, atopic dermatitis, autoimmune dermatitis, autoimmune disorder associated with streptococcus infection, autoimmune enteropathy, autoimmune hearing loss, autoimmune lymphoproliferative syndrome (ALPS), autoimmune myocarditis, autoimmune premature ovarian failure, blepharitis, bronchiectasis, bullous pemphigoid, cardiovascular disease, catastrophic antiphospholipid syndrome, celiac disease, cervical spondylosis, chronic ischemia, cicatricial pemphigoid, clinically isolated syndrome (CIS) with risk for multiple sclerosis, conjunctivitis, childhood onset psychiatric disorder, chronic obstructive pulmonary disease (COPD), dacryocystitis, dermatomyositis, diabetic retinopathy, diabetes mellitus, disk herniation, disk prolaps, drug induced immune hemolytic anemia, endocarditis, endometriosis, endophthalmitis, episcleritis, erythema multiforme, erythema multiforme major, gestational pemphigoid, Guillain-Barré syndrome (GBS), hay fever, Hughes syndrome, idiopathic Parkinson's disease, idiopathic interstitial pneumonia, IgE-mediated allergy, immune hemolytic anemia, inclusion body myositis, infectious ocular inflammatory disease, inflammatory demyelinating disease, inflammatory heart disease, inflammatory kidney disease, IPF/UIP, iritis, keratitis, keratojuntivitis sicca, Kussmaul disease or Kussmaul-Meier disease, Landry's paralysis, Langerhan's cell histiocytosis, livedo reticularis, macular degeneration, microscopic polyangiitis, morbus bechterev, motor neuron disorders, mucous membrane pemphigoid, multiple organ failure, myasthenia gravis, myelodysplastic syndrome, myocarditis, nerve root disorders, neuropathy, non-A non-B hepatitis, optic neuritis, osteolysis, ovarian cancer, pauciarticular JRA, peripheral artery occlusive disease (PAOD), peripheral vascular disease (PVD), peripheral artery, disease (PAD), phlebitis, polyarteritis nodosa (or periarteritis nodosa), polychondritis, polymyalgia rheumatica, polio sis, polyarticular JRA, polyendocrine deficiency syndrome, polymyositis, polymyalgia rheumatica (PMR), post-pump syndrome, primary Parkinsonism, prostate and rectal cancer and hematopoietic malignancies (leukemia and lymphoma), prostatitis, pure red cell aplasia, primary adrenal insufficiency, recurrent neuromyelitis optica, restenosis, rheumatic heart disease, sapho (synovitis, acne, pustulosis, hyperostosis, and osteitis), scleroderma, secondary amyloidosis, shock lung, scleritis, sciatica, secondary adrenal insufficiency, silicone associated connective tissue disease, sneddon-wilkinson dermatosis, spondilitis ankylosans, Stevens-Johnson syndrome (SJS), systemic inflammatory response syndrome, temporal arteritis, toxoplasmic retinitis, toxic epidermal necrolysis, transverse myelitis, TRAPS (tumor necrosis factor receptor, type 1 allergic reaction, type II diabetes, urticaria, usual interstitial pneumonia (UIP), vasculitis, vernal conjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome (VKH syndrome), wet macular degeneration (wet AMD), dry macular degeneration (dry AMD) and other inflammatory disorders of the eye (e.g., uveitis, retinitis, choroiditis, sclerosis, dry eye syndrome (DES) and the like), wound healing, yersinia and salmonella associated arthropathy.

In an embodiment, the compositions and methods described herein can be used to treat pain in an individual suffering from a disease selected from the group consisting of primary and metastatic cancers, including carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gallbladder and bile ducts, small intestine, urinary tract (including kidney, bladder and urothelium), female genital tract (including cervix, uterus, and ovaries as well as choriocarcinoma and gestational trophoblastic disease), male genital tract (including prostate, seminal vesicles, testes and germ cell tumors), endocrine glands (including the thyroid, adrenal, and pituitary glands), and skin, as well as hemangiomas, melanomas, sarcomas (including those arising from bone and soft tissues as well as Kaposi's sarcoma), tumors of the brain, nerves, eyes, and meninges (including astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas, and meningiomas), solid tumors arising from hematopoietic malignancies such as leukemias, and lymphomas (both Hodgkin's and non-Hodgkin's lymphomas).

IV. Pharmaceutical Compositions

The disclosure also provides pharmaceutical compositions comprising binding proteins (including DVD-Igs described herein), of the disclosure and a pharmaceutically acceptable carrier. The pharmaceutical compositions comprising antibodies of the disclosure are for use in, but not limited to, diagnosing, detecting, or monitoring a disorder, in preventing, treating, managing, or ameliorating of a disorder or one or more symptoms thereof, and/or in research. In a specific embodiment, a composition comprises one or more antibodies of the disclosure. In another embodiment, the pharmaceutical composition comprises one or more antibodies of the disclosure and one or more prophylactic or therapeutic agents other than antibodies of the disclosure for treating a disorder in which IL-1α and or β activity is detrimental. In an embodiment, the prophylactic or therapeutic agents are known to be useful for or having been or currently being used in the prevention, treatment, management, or amelioration of a disorder or one or more symptoms thereof. In accordance with these embodiments, the composition may further comprise of a carrier, diluent or excipient.

The binding proteins of the disclosure can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises a binding protein of the disclosure and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the binding protein.

Various delivery systems are known and can be used to administer one or more antibodies of the disclosure or the combination of one or more antibodies of the disclosure and a prophylactic agent or therapeutic agent useful for preventing, managing, treating, or ameliorating a disorder or one or more symptoms thereof, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the binding protein, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem., 262: 4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector. Methods of administering a prophylactic or therapeutic agent of the disclosure include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural administration, intratumoral administration, and mucosal administration (e.g., intranasal and oral routes). In addition, pulmonary administration can be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064; 5,855,913 and 5,290,540; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated herein by reference their entireties. In one embodiment, a binding protein of the disclosure, combination therapy, or a composition of the disclosure is administered using Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.). In a specific embodiment, prophylactic or therapeutic agents of the disclosure are administered intramuscularly, intravenously, intratumorally, orally, intranasally, pulmonary, or subcutaneously. The prophylactic or therapeutic agents may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.

In an embodiment, specific binding of antibody-coupled carbon nanotubes (CNTs) to tumor cells in vitro, followed by their highly specific ablation with near-infrared (NIR) light can be used to target tumor cells. For example, biotinylated polar lipids can be used to prepare stable, biocompatible, noncytotoxic CNT dispersions that are then attached to one or two different neutralite avidin-derivatized DVD-Igs directed against one or more tumor antigens (e.g., CD22) (Chakravarty et al., Proc. Natl. Acad. Sci. USA, 105: 8697-8702 (2008)).

In a specific embodiment, it may be desirable to administer the prophylactic or therapeutic agents of the disclosure locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous or non-porous material, including membranes and matrices, such as sialastic membranes, polymers, fibrous matrices (e.g., Tissuel®), or collagen matrices. In one embodiment, an effective amount of one or more antibodies of the disclosure antagonists is administered locally to the affected area to a subject to prevent, treat, manage, and/or ameliorate a disorder or a symptom thereof. In another embodiment, an effective amount of one or more antibodies of the disclosure is administered locally to the affected area in combination with an effective amount of one or more therapies (e.g., one or more prophylactic or therapeutic agents) other than a binding protein of the disclosure of a subject to prevent, treat, manage, and/or ameliorate a disorder or one or more symptoms thereof.

In another embodiment, the prophylactic or therapeutic agent can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release (see Langer, supra; Sefton, M. V., CRC Crit. Rev. Biomed. Eng., 14: 201-240 (1987); Buchwald et al., Surgery, 88: 507-516 (1980); Saudek et al., N. Engl. J. Med., 321: 574-579 (1989)). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the therapies of the disclosure (see, e.g., Goodson, J. M., Chapter 6, In Medical Applications of Controlled Release, Vol. II, Applications and Evaluation, (Langer and Wise, eds.) (CRC Press, Inc., Boca Raton, 1984) pp. 115-138; Langer and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. Phys., C23(1): 61-126 (1983); see also Levy et al., Science, 228:190-192 (1985); During et al., Ann. Neurol., 25:351-356 (1989); Howard et al., J. Neurosurg., 71:105-112 (1989)); U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253. Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In an exemplary embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In yet another embodiment, a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Controlled release systems are discussed in the review by Langer (Science, 249:1527-1533 (1990)). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more therapeutic agents of the disclosure. See, e.g., U.S. Pat. No. 4,526,938, PCT Publication No. WO 91/05548, PCT Publication No. WO 96/20698; Ning et al., “Intratumoral radioimmunotherapy of a human colon cancer xenograft using a sustained-release gel,” Radiotherapy Oncol., 39: 179-189 (1996); Song et al., “Antibody Mediated Lung Targeting of Long-Circulating Emulsions,” PDA J. Pharm. Sci. Technol., 50: 372-377 (1996); Cleek et al., “Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application,” Proceed. Int'l. Symp. Control. Rel. Bioact. Mater., 24: 853-854 (1997); and Lam et al., “Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery,” Proceed. Int'l. Symp. Control Rel. Bioact. Mater., 24: 759-760 (1997), each of which is incorporated herein by reference in their entireties.

In a specific embodiment, where the composition of the disclosure is a nucleic acid encoding a prophylactic or therapeutic agent, the nucleic acid can be administered in vivo to promote expression of its encoded prophylactic or therapeutic agent, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic®, DuPont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see, e.g., Joliot et al., Proc. Natl. Acad. Sci. USA, 88: 1864-1868 (1991)). Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression by homologous recombination.

A pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the site of the injection.

If the compositions of the disclosure are to be administered topically, the compositions can be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity preferably greater than water are typically employed. Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as FREON®) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well known in the art.

If the method of the disclosure comprises intranasal administration of a composition, the composition can be formulated in an aerosol form, spray, mist or in the form of drops. In particular, prophylactic or therapeutic agents for use according to the present disclosure can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, e.g., gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

If the method of the disclosure comprises oral administration, compositions can be formulated orally in the form of tablets, capsules, cachets, gelcaps, solutions, suspensions, and the like. Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well-known in the art. Liquid preparations for oral administration may take the form of, but not limited to, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of a prophylactic or therapeutic agent(s).

The method of the disclosure may comprise pulmonary administration, e.g., by use of an inhaler or nebulizer, of a composition formulated with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064; 5,855,913; and 5,290,540; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated herein by reference their entireties. In a specific embodiment, a binding protein of the disclosure, combination therapy, and/or composition of the disclosure is administered using Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).

The method of the disclosure may comprise administration of a composition formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.

The methods of the disclosure may additionally comprise of administration of compositions formulated as depot preparations. Such long acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

The methods of the disclosure encompass administration of compositions formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

Generally, the ingredients of compositions are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the mode of administration is infusion, composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the mode of administration is by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

In particular, the disclosure also provides that one or more of the prophylactic or therapeutic agents, or pharmaceutical compositions of the disclosure is packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the agent. In one embodiment, one or more of the prophylactic or therapeutic agents, or pharmaceutical compositions of the disclosure is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. Preferably, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions of the disclosure is supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg, at least 100 mg or at least 200 mg/mL. The lyophilized prophylactic or therapeutic agents or pharmaceutical compositions of the disclosure should be stored at between 2° C. and 8° C. in its original container and the prophylactic or therapeutic agents, or pharmaceutical compositions of the disclosure should be administered within 1 week, preferably within 5 days, within 72 hours, within 48 hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted. In an alternative embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions of the disclosure is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the agent. Preferably, the liquid form of the administered composition is supplied in a hermetically sealed container at least 0.25 mg/ml, more preferably at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, at least 75 mg/ml, at least 100 mg/ml or at least 200 mg/mL. The liquid form should be stored at between 2° C. and 8° C. in its original container.

The binding protein of the disclosure can be incorporated into a pharmaceutical composition suitable for parenteral administration. Preferably, the binding protein will be prepared as an injectable solution containing 0.1-250 mg/ml binding protein. The injectable solution can be composed of either a liquid or lyophilized dosage form in a flint or amber vial, ampoule or pre-filled syringe. The buffer can be L-histidine (1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium chloride can be used to modify the toxicity of the solution at a concentration of 0-300 mM (optimally 150 mM for a liquid dosage form). Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Bulking agents can be included for a lyophilized dosage form, principally 1-10% mannitol (optimally 2-4%). Stabilizers can be used in both liquid and lyophilized dosage forms, principally 1-50 mM L-Methionine (optimally 5-10 mM). Other suitable bulking agents include glycine, arginine, can be included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%). Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants. The pharmaceutical composition comprising a binding protein of the disclosure prepared as an injectable solution for parenteral administration, can further comprise an agent useful as an adjuvant, such as those used to increase the absorption, or dispersion of a therapeutic protein (e.g., DVD-Ig). A particularly useful adjuvant is hyaluronidase (such as Hylenex® recombinant human hyaluronidase). Addition of hyaluronidase in the injectable solution improves human bioavailability following parenteral administration, particularly subcutaneous administration. It also allows for greater injection site volumes (i.e., greater than 1 ml) with less pain and discomfort, and minimum incidence of injection site reactions (see, PCT Publication No. WO 2004/078140 and US Publication No. 2006/104968).

The compositions provided in this disclosure may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In an exemplary embodiment, the binding protein is administered by intravenous infusion or injection. In another preferred embodiment, the binding protein is administered by intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., binding protein) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile, lyophilized powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and spray-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including, in the composition, an agent that delays absorption, for example, monostearate salts and gelatin.

The binding proteins of the present disclosure can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is subcutaneous injection, intravenous injection or infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, (J. R. Robinson, ed.) (Marcel Dekker, Inc., New York, 1978).

In certain embodiments, a binding protein of the disclosure may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the disclosure by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.

Supplementary active compounds can also be incorporated into the compositions. In certain embodiments, a binding protein of the disclosure is coformulated with and/or coadministered with one or more additional therapeutic agents that are useful for treating disorders in which IL-1α and/or β activity is detrimental. For example, an anti-human IL-1α or β binding protein of the disclosure may be coformulated and/or coadministered with one or more additional antibodies that bind other targets (e.g., antibodies that bind other cytokines or that bind cell surface molecules). Furthermore, one or more antibodies of the disclosure may be used in combination with two or more of the foregoing therapeutic agents. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.

In certain embodiments, a binding protein to IL-1α or β or fragment thereof is linked to a half-life extending vehicle known in the art. Such vehicles include, but are not limited to, the Fc domain, polyethylene glycol, and dextran. Such vehicles are described, e.g., in U.S. Ser. No. 09/428,082 (now U.S. Pat. No. 6,660,843) which is hereby incorporated by reference for any purpose.

In a specific embodiment, nucleic acid sequences comprising nucleotide sequences encoding a binding protein of the disclosure or another prophylactic or therapeutic agent of the disclosure are administered to treat, prevent, manage, or ameliorate a disorder or one or more symptoms thereof by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the disclosure, the nucleic acids produce their encoded binding protein or prophylactic or therapeutic agent of the disclosure that mediates a prophylactic or therapeutic effect.

Any of the methods for gene therapy available in the art can be used according to the present disclosure. For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharm., 12: 488-505 (1993); Wu et al., “Delivery systems for gene therapy,” Biotherapy, 3: 87-95 (1991); Tolstoshev, P., Ann. Rev. Pharmacol. Toxicol., 32: 573-596 (1993); Mulligan, R. C., Science, 260: 926-932 (1993); and Morgan and Anderson, “Human Gene Therapy,” Ann. Rev. Biochem., 62:191-217 (1993); Robinson, C., Trends Biotechnol., 11:155 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, New York (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, New York (1990). Detailed description of various methods of gene therapy are disclosed in US Publication No. 2005/0042664 A1, which is incorporated herein by reference.

A binding protein of the disclosure also can be administered with one or more additional therapeutic agents useful in the treatment of various diseases. Binding proteins of the disclosure, can be used alone or in combination to treat such diseases. It should be understood that the binding proteins of the disclosure can be used alone or in combination with an additional agent, e.g., a therapeutic agent, said additional agent being selected by the skilled artisan for its intended purpose. For example, the additional agent can be a therapeutic agent art-recognized as being useful to treat the disease or condition being treated by the binding protein of the present disclosure. The additional agent also can be an agent that imparts a beneficial attribute to the therapeutic composition, e.g., an agent that affects the viscosity of the composition.

It should further be understood that the combinations which are to be included within this disclosure are those combinations useful for their intended purpose. The agents set forth below are illustrative for purposes and not intended to be limited. The combinations, which are part of this disclosure, can be the antibodies of the present disclosure and at least one additional agent selected from the lists below. The combination can also include more than one additional agent, e.g., two or three additional agents if the combination is such that the formed composition can perform its intended function.

Preferred combinations are non-steroidal anti-inflammatory drug(s) also referred to as NSAIDS which include drugs like ibuprofen. Other preferred combinations are corticosteroids including prednisolone; the well known side-effects of steroid use can be reduced or even eliminated by tapering the steroid dose required when treating patients in combination with the anti-IL-1α and β binding proteins of this disclosure. Non-limiting examples of therapeutic agents for rheumatoid arthritis with which a binding protein can be combined include, but are not limited to, the following: cytokine suppressive anti-inflammatory drug(s) (CSAIDs); antibodies to or antagonists of other human cytokines or growth factors, for example, TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, IL-21, interferons, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the disclosure, or antigen binding portions thereof, can be combined with antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA or their ligands including CD154 (gp39 or CD40L).

Preferred combinations of therapeutic agents may interfere at different points in the autoimmune and subsequent inflammatory cascade; preferred examples include TNF antagonists like chimeric, humanized or human TNF antibodies, D2E7, (PCT Publication No. WO 97/29131), CA2 (Remicade™), CDP 571, and soluble p55 or p75 TNF receptors, derivatives, thereof, (p75TNFR1gG (Enbrel™) or p55TNFR1gG (Lenercept), and also TNFα converting enzyme (TACE) inhibitors; similarly IL-1 inhibitors (Interleukin-1-converting enzyme inhibitors, IL-1RA etc.) may be effective for the same reason. Other preferred combinations include Interleukin 11. Yet another preferred combination are other key players of the autoimmune response which may act parallel to, dependent on or in concert with IL-1α or β function. Yet another preferred combination are non-depleting anti-CD4 inhibitors. Yet other preferred combinations include antagonists of the co-stimulatory pathway CD80 (B7.1) or CD86 (B7.2) including antibodies, soluble receptors or antagonistic ligands.

The binding proteins of the disclosure may also be combined with agents, such as methotrexate, 6-MP, azathioprine sulphasalazine, mesalazine, olsalazine chloroquinine/hydroxychloroquine, pencillamine, aurothiomalate (intramuscular and oral), azathioprine, colchicine, corticosteroids (oral, inhaled and local injection), beta-2 adrenoreceptor agonists (salbutamol, terbutaline, salmeteral), xanthines (theophylline, aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium and oxitropium, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adensosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, agents which interfere with signaling by proinflammatory cytokines such as TNF-α or IL-1 (e.g., IRAK, NIK, IKK, p38, or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TNFα converting enzyme (TACE) inhibitors, T-cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof (e.g., soluble p55 or p75 TNF receptors and the derivatives p75TNFRIgG (Enbrel™ and p55TNFRIgG (Lenercept)), sIL-1RI, sIL-1RII, sIL-6R), anti-inflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13 and TGFβ), celecoxib, folic acid, hydroxychloroquine sulfate, rofecoxib, etanercept, infliximab, naproxen, valdecoxib, sulfasalazine, methylprednisolone, meloxicam, methylprednisolone acetate, gold sodium thiomalate, aspirin, triamcinolone acetonide, propoxyphene napsylate/apap, folate, nabumetone, diclofenac, piroxicam, etodolac, diclofenac sodium, oxaprozin, oxycodone HCl, hydrocodone bitartrate/apap, diclofenac sodium/misoprostol, fentanyl, anakinra, human recombinant, tramadol HCl, salsalate, sulindac, cyanocobalamin/fa/pyridoxine, acetaminophen, alendronate sodium, prednisolone, morphine sulfate, lidocaine hydrochloride, indomethacin, glucosamine sulf/chondroitin, amitriptyline HCl, sulfadiazine, oxycodone HCl/acetaminophen, olopatadine HCl, misoprostol, naproxen sodium, omeprazole, cyclophosphamide, rituximab, IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18, anti-IL15, BIRB-796, SCIO-469, VX-702, AMG-548, VX-740, Roflumilast, IC-485, CDC-801, and Mesopram. Preferred combinations include methotrexate or leflunomide and in moderate or severe rheumatoid arthritis cases, cyclosporine.

Non-limiting additional agents which can also be used in combination with a binding protein to treat rheumatoid arthritis (RA) include, but are not limited to, the following: non-steroidal anti-inflammatory drug(s) (NSAIDs); cytokine suppressive anti-inflammatory drug(s) (CSAIDs); CDP-571/BAY-10-3356 (humanized anti-TNFα antibody; Celltech/Bayer); cA2/infliximab (chimeric anti-TNFα antibody; Centocor); 75 kdTNFR-IgG/etanercept (75 kD TNF receptor-IgG fusion protein; Immunex; see e.g., Moreland et al. (Abstract No. 813), Arthritis Rheum., 37: 5295 (1994); Baumgartner et al., J. Invest. Med., 44(3): 235A (March 1996); 55 kdTNF-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (non-depleting primatized anti-CD4 antibody; IDEC/SmithKline; see e.g., Kaine et al. (Abstract No. 195), Arthritis Rheum., 38: 5185 (1995)); DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2 fusion proteins; Seragen; see e.g., Sewell et al., Arthritis Rheum., 36(9): 1223-1233 (September 1993)); Anti-Tac (humanized anti-IL-2Rα; Protein Design Labs/Roche); IL-4 (anti-inflammatory cytokine; DNAX/Schering); IL-10 (SCH 52000; recombinant IL-10, anti-inflammatory cytokine; DNAX/Schering); IL-4; IL-10 and/or IL-4 agonists (e.g., agonist antibodies); IL-1RA (IL-1 receptor antagonist; Synergen/Amgen); anakinra (Kineret®/Amgen); TNF-bp/s-TNF (soluble TNF binding protein; see e.g., Evans et al. (Abstract No. 1540), Arthritis Rheum., 39(9)(supplement): 5284 (1996)); Kapadia et al., Amer. J. Physiol.—Heart and Circulatory Physiology, 268: H517-H525 (1995)); RP73401 (phosphodiesterase Type IV inhibitor; see e.g., Chikanza et al. (Abstract No. 1527), Arthritis Rheum., 39(9)(supplement): 5282 (1996)); MK-966 (COX-2 Inhibitor; see e.g., Erich et al. (Abstract Nos. 328 and 329), Arthritis Rheum., 39(9)(supplement): S81 (1996)); Iloprost (see e.g., Scholz, P. (Abstract No. 336), Arthritis Rheum., 39(9)(supplement): S82 (1996)); methotrexate; thalidomide (see e.g., Lee et al. (Abstract No. 1524), Arthritis Rheum., 39(9)(supplement): 5282 (1996)) and thalidomide-related drugs (e.g., Celgen); leflunomide (anti-inflammatory and cytokine inhibitor; see e.g., Finnegan et al. (Abstract No. 627), Arthritis Rheum., 39(9)(supplement): S131 (1996)); Thoss et al., Inflamm. Res., 45: 103-107 (1996)); tranexamic acid (inhibitor of plasminogen activation; see e.g., Ronday et al. (Abstract No. 1541), Arthritis Rheum., 39(9)(supplement): 5284 (1996)); T-614 (cytokine inhibitor; see e.g., Hara et al. (Abstract No. 1526), Arthritis Rheum., 39(9)(supplement): 5282 (1996)); prostaglandin E1 (see e.g., Moriuchi et al. (Abstract No. 1528), Arthritis Rheum., 39(9)(supplement): 5282 (1996)); Tenidap (non-steroidal anti-inflammatory drug; see e.g., Guttadauria, M. (Abstract No. 1516), Arthritis Rheum., 39(9)(supplement): 5280 (1996)); Naproxen (non-steroidal anti-inflammatory drug; see e.g., Fiebich et al., Neuro Report, 7: 1209-1213 (1996)); Meloxicam (non-steroidal anti-inflammatory drug); Ibuprofen (non-steroidal anti-inflammatory drug); Piroxicam (non-steroidal anti-inflammatory drug); Diclofenac (non-steroidal anti-inflammatory drug); Indomethacin (non-steroidal anti-inflammatory drug); Sulfasalazine (see e.g., Farr et al. (Abstract No. 1519), Arthritis Rheum., 39(9)(supplement): S281 (1996)); Azathioprine (see e.g., Hickey et al. (Abstract No. 1521), Arthritis Rheum., 39(9)(supplement): S281 (1996)); ICE inhibitor (inhibitor of the enzyme interleukin-1β converting enzyme); zap-70 and/or lck inhibitor (inhibitor of the tyrosine kinase zap-70 or lck); VEGF inhibitor and/or VEGF-R inhibitor (inhibitors of vascular endothelial cell growth factor or vascular endothelial cell growth factor receptor; inhibitors of angiogenesis); cortico steroid anti-inflammatory drugs (e.g., SB203580); TNF-convertase inhibitors; anti-IL-12 antibodies; anti-IL-18 antibodies; interleukin-11 (see e.g., Keith Jr. et al. (Abstract No. 1613), Arthritis Rheum., 39(9)(supplement): S296 (1996)); interleukin-13 (see e.g., Bessis et al. (Abstract No. 1681), Arthritis Rheum., 39(9)(supplement): 5308 (1996)); interleukin-17 inhibitors (see e.g., Lotz et al. (Abstract No. 559), Arthritis Rheum., 39(9)(supplement): 5120 (1996)); gold; penicillamine; chloroquine; chlorambucil; hydroxychloroquine; cyclosporine; cyclophosphamide; total lymphoid irradiation; anti-thymocyte globulin; anti-CD4 antibodies; CD5-toxins; orally-administered peptides and collagen; lobenzarit disodium; Cytokine Regulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisense phosphorothioate oligo-deoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycan polysulphate; minocycline; anti-IL2R antibodies; marine and botanical lipids (fish and plant seed fatty acids; see e.g., DeLuca et al., Rheum. Dis. Clin. North Am., 21: 759-777 (1995)); auranofin; phenylbutazone; meclofenamic acid; flufenamic acid; intravenous immune globulin; zileuton; azaribine; mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); amiprilose (therafectin); cladribine (2-chlorodeoxyadenosine); methotrexate; bcl-2 inhibitors (see Bruncko et al., J. Med. Chem., 50(4), 641-662 (2007)); antivirals and immune modulating agents.

In one embodiment, the binding protein described herein is administered in combination with one of the following agents for the treatment of rheumatoid arthritis (RA): small molecule inhibitor of KDR, small molecule inhibitor of Tie-2; methotrexate; prednisone; celecoxib; folic acid; hydroxychloroquine sulfate; rofecoxib; etanercept; infliximab; leflunomide; naproxen; valdecoxib; sulfasalazine; methylprednisolone; ibuprofen; meloxicam; methylprednisolone acetate; gold sodium thiomalate; aspirin; azathioprine; triamcinolone acetonide; propoxyphene napsylate/apap; folate; nabumetone; diclofenac; piroxicam; etodolac; diclofenac sodium; oxaprozin; oxycodone HCl; hydrocodone bitartrate/apap; diclofenac sodium/misoprostol; fentanyl; anakinra, human recombinant; tramadol HCl; salsalate; sulindac; cyanocobalamin/fa/pyridoxine; acetaminophen; alendronate sodium; prednisolone; morphine sulfate; lidocaine hydrochloride; indomethacin; glucosamine sulfate/chondroitin; cyclosporine; amitriptyline HCl; sulfadiazine; oxycodone HCl/acetaminophen; olopatadine HCl; misoprostol; naproxen sodium; omeprazole; mycophenolate mofetil; cyclophosphamide; rituximab; IL-1 TRAP; MRA; CTLA4-IG; IL-18 BP; IL-12/23; anti-IL 18; anti-IL 15; BIRB-796; SCIO-469; VX-702; AMG-548; VX-740; Roflumilast; IC-485; CDC-801; and mesopram.

Non-limiting examples of therapeutic agents for inflammatory bowel disease with which a binding protein of the disclosure can be combined include the following: budenoside; epidermal growth factor; corticosteroids; cyclosporin, sulfasalazine; amino salicylates; 6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor antagonists; anti-IL-1β mAbs; anti-IL-6 mAbs; growth factors; elastase inhibitors; pyridinyl-imidazole compounds; antibodies to or antagonists of other human cytokines or growth factors, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-17, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the disclosure, or antigen binding portions thereof, can be combined with antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands. The binding proteins of the disclosure may also be combined with agents, such as methotrexate, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, agents which interfere with signaling by proinflammatory cytokines such as TNFα or IL-1 (e.g., IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TNFα converting enzyme inhibitors, T-cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof (e.g., soluble p55 or p75 TNF receptors, sIL-1RI, sIL-1RII, sIL-6R) and anti-inflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13 and TGFβ) and bcl-2 inhibitors.

Non-limiting examples of therapeutic agents for multiple sclerosis (MS) with which binding proteins of the disclosure can be combined include the following: corticosteroids; prednisolone; methylprednisolone; azathioprine; cyclophosphamide; cyclosporine; methotrexate; 4-aminopyridine; tizanidine; interferon-β1a (AVONEX; Biogen); interferon-β1b (BETASERON; Chiron/Berlex); interferon α-n3) (Interferon Sciences/Fujimoto), interferon-α1b (Alfa Wassermann/J&J), interferon β1A-IF (Serono/Inhale Therapeutics), Peginterferon α 2b (Enzon/Schering-Plough), Copolymer 1 (Cop-1; COPAXONE; Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous immunoglobulin; clabribine; antibodies to or antagonists of other human cytokines or growth factors and their receptors, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-23, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Binding proteins of the disclosure can be combined with antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 or their ligands. Binding proteins of the disclosure, may also be combined with agents, such as methotrexate, cyclosporine, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adensosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, agents which interfere with signaling by proinflammatory cytokines such as TNFα or IL-1 (e.g., IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TACE inhibitors, T-cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof (e.g., soluble p55 or p75 TNF receptors, sIL-1RI, sIL-1RII, sIL-6R), anti-inflammatory cytokines (e.g., IL-4, IL-1β, IL-13 and TGFβ) and bcl-2 inhibitors.

Examples of therapeutic agents for multiple sclerosis with which binding proteins of the disclosure can be combined include interferon-β, for example, IFNβ1a and IFNβ1b; copaxone; corticosteroids; caspase inhibitors, for example inhibitors of caspase-1; IL-1 inhibitors; TNF inhibitors; and antibodies to CD40 ligand and CD80.

The binding proteins of the disclosure, may also be combined with agents, such as alemtuzumab, dronabinol, Unimed, daclizumab, mitoxantrone, xaliproden hydrochloride, fampridine, glatiramer acetate, natalizumab, sinnabidol, a-immunokine NNSO3, ABR-215062, AnergiX.MS, chemokine receptor antagonists, BBR-2778, calagualine, CPI-1189, LEM (liposome encapsulated mitoxantrone), THC.CBD (cannabinoid agonist) MBP-8298, mesopram (PDE4 inhibitor), MNA-715, anti-IL-6 receptor antibody, neurovax, pirfenidone allotrap 1258 (RDP-1258), sTNF-R1, talampanel, teriflunomide, TGF-beta2, tiplimotide, VLA-4 antagonists (for example, TR-14035, VLA4 Ultrahaler, Antegran-ELAN/Biogen), interferon gamma antagonists, IL-4 agonists.

Non-limiting examples of therapeutic agents for angina with which binding proteins of the disclosure can be combined include the following: aspirin, nitroglycerin, isosorbide mononitrate, metoprolol succinate, atenolol, metoprolol tartrate, amlodipine besylate, diltiazem hydrochloride, isosorbide dinitrate, clopidogrel bisulfate, nifedipine, atorvastatin calcium, potassium chloride, furosemide, simvastatin, verapamil HCl, digoxin, propranolol hydrochloride, carvedilol, lisinopril, spironolactone, hydrochlorothiazide, enalapril maleate, nadolol, ramipril, enoxaparin sodium, heparin sodium, valsartan, sotalol hydrochloride, fenofibrate, ezetimibe, bumetanide, losartan potassium, lisinopril/hydrochlorothiazide, felodipine, captopril, bisoprolol fumarate.

Non-limiting examples of therapeutic agents for ankylosing spondylitis with which binding proteins of the disclosure can be combined include the following: ibuprofen, diclofenac and misoprostol, naproxen, meloxicam, indomethacin, diclofenac, celecoxib, rofecoxib, sulfasalazine, methotrexate, azathioprine, minocyclin, prednisone, etanercept, infliximab.

Non-limiting examples of therapeutic agents for asthma with which binding proteins of the disclosure can be combined include the following: albuterol, salmeterol/fluticasone, montelukast sodium, fluticasone propionate, budesonide, prednisone, salmeterol xinafoate, levalbuterol HCl, albuterol sulfate/ipratropium, prednisolone sodium phosphate, triamcinolone acetonide, beclomethasone dipropionate, ipratropium bromide, azithromycin, pirbuterol acetate, prednisolone, theophylline anhydrous, methylprednisolone sodium succinate, clarithromycin, zafirlukast, formoterol fumarate, influenza virus vaccine, methylprednisolone, amoxicillin trihydrate, flunisolide, allergy injection, cromolyn sodium, fexofenadine hydrochloride, flunisolide/menthol, amoxicillin/clavulanate, levofloxacin, inhaler assist device, guaifenesin, dexamethasone sodium phosphate, moxifloxacin HCl, doxycycline hyclate, guaifenesin/d-methorphan, p-ephedrine/cod/chlorphenir, gatifloxacin, cetirizine hydrochloride, mometasone furoate, salmeterol xinafoate, benzonatate, cephalexin, pe/hydrocodone/chlorphenir, cetirizine HCl/pseudoephed, phenylephrine/cod/promethazine, codeine/promethazine, cefprozil, dexamethasone, guaifenesin/pseudoephedrine, chlorpheniramine/hydrocodone, nedocromil sodium, terbutaline sulfate, epinephrine, methylprednisolone, metaproterenol sulfate.

Non-limiting examples of therapeutic agents for COPD with which binding proteins of the disclosure can be combined include the following: albuterol sulfate/ipratropium, ipratropium bromide, salmeterol/fluticasone, albuterol, salmeterol xinafoate, fluticasone propionate, prednisone, theophylline anhydrous, methylprednisolone sodium succinate, montelukast sodium, budesonide, formoterol fumarate, triamcinolone acetonide, levofloxacin, guaifenesin, azithromycin, beclomethasone dipropionate, levalbuterol HCl, flunisolide, ceftriaxone sodium, amoxicillin trihydrate, gatifloxacin, zafirlukast, amoxicillin/clavulanate, flunisolide/menthol, chlorpheniramine/hydrocodone, metaproterenol sulfate, methylprednisolone, mometasone furoate, p-ephedrine/cod/chlorphenir, pirbuterol acetate, p-ephedrine/loratadine, terbutaline sulfate, tiotropium bromide, (R,R)-formoterol, TgAAT, Cilomilast, Roflumilast.

Non-limiting examples of therapeutic agents for HCV with which binding proteins of the disclosure can be combined include the following: Interferon-alpha-2a, Interferon-alpha-2b, Interferon-alpha con1, Interferon-alpha-n1, PEGylated interferon-alpha-2a, PEGylated interferon-alpha-2b, ribavirin, Peginterferon alfa-2b+ribavirin, Ursodeoxycholic Acid, Glycyrrhizic Acid, Thymalfasin, Maxamine, VX-497 and any compounds that are used to treat HCV through intervention with the following targets: HCV polymerase, HCV protease, HCV helicase, HCV IRES (internal ribosome entry site).

Non-limiting examples of therapeutic agents for idiopathic pulmonary fibrosis with which binding proteins of the disclosure can be combined include the following: prednisone, azathioprine, albuterol, colchicine, albuterol sulfate, digoxin, gamma interferon, methylprednisolone sod succ, lorazepam, furosemide, lisinopril, nitroglycerin, spironolactone, cyclophosphamide, ipratropium bromide, actinomycin d, alteplase, fluticasone propionate, levofloxacin, metaproterenol sulfate, morphine sulfate, oxycodone hcl, potassium chloride, triamcinolone acetonide, tacrolimus anhydrous, calcium, interferon-alpha, methotrexate, mycophenolate mofetil, Interferon-gamma-1β.

Non-limiting examples of therapeutic agents for myocardial infarction with which binding proteins of the disclosure can be combined include the following: aspirin, nitroglycerin, metoprolol tartrate, enoxaparin sodium, heparin sodium, clopidogrel bisulfate, carvedilol, atenolol, morphine sulfate, metoprolol succinate, warfarin sodium, lisinopril, isosorbide mononitrate, digoxin, furosemide, simvastatin, ramipril, tenecteplase, enalapril maleate, torsemide, retavase, losartan potassium, quinapril HCl/mag carb, bumetanide, alteplase, enalaprilat, amiodarone hydrochloride, tirofiban HCl m-hydrate, diltiazem hydrochloride, captopril, irbesartan, valsartan, propranolol hydrochloride, fosinopril sodium, lidocaine hydrochloride, eptifibatide, cefazolin sodium, atropine sulfate, aminocaproic acid, spironolactone, interferon, sotalol hydrochloride, potassium chloride, docusate sodium, dobutamine HCl, alprazolam, pravastatin sodium, atorvastatin calcium, midazolam hydrochloride, meperidine hydrochloride, isosorbide dinitrate, epinephrine, dopamine hydrochloride, bivalirudin, rosuvastatin, ezetimibe/simvastatin, avasimibe, cariporide.

Non-limiting examples of therapeutic agents for psoriasis with which binding proteins of the disclosure can be combined include the following: small molecule inhibitor of KDR, small molecule inhibitor of Tie-2, calcipotriene, clobetasol propionate, triamcinolone acetonide, halobetasol propionate, tazarotene, methotrexate, fluocinonide, betamethasone diprop augmented, fluocinolone acetonide, acitretin, tar shampoo, betamethasone valerate, mometasone furoate, ketoconazole, pramoxine/fluocinolone, hydrocortisone valerate, flurandrenolide, urea, betamethasone, clobetasol propionate/emoll, fluticasone propionate, azithromycin, hydrocortisone, moisturizing formula, folic acid, desonide, pimecrolimus, coal tar, diflorasone diacetate, etanercept folate, lactic acid, methoxsalen, hc/bismuth subgal/znox/resor, methylprednisolone acetate, prednisone, sunscreen, halcinonide, salicylic acid, anthralin, clocortolone pivalate, coal extract, coal tar/salicylic acid, coal tar/salicylic acid/sulfur, desoximetasone, diazepam, emollient, fluocinonide/emollient, mineral oil/castor oil/na lact, mineral oil/peanut oil, petroleum/isopropyl myristate, psoralen, salicylic acid, soap/tribromsalan, thimerosal/boric acid, celecoxib, infliximab, cyclosporine, alefacept, efalizumab, tacrolimus, pimecrolimus, PUVA, UVB, sulfasalazine.

Non-limiting examples of therapeutic agents for psoriatic arthritis with which binding proteins of the disclosure can be combined include the following: methotrexate, etanercept, rofecoxib, celecoxib, folic acid, sulfasalazine, naproxen, leflunomide, methylprednisolone acetate, indomethacin, hydroxychloroquine sulfate, prednisone, sulindac, betamethasone diprop augmented, infliximab, methotrexate, folate, triamcinolone acetonide, diclofenac, dimethylsulfoxide, piroxicam, diclofenac sodium, ketoprofen, meloxicam, methylprednisolone, nabumetone, tolmetin sodium, calcipotriene, cyclosporine, diclofenac sodium/misoprostol, fluocinonide, glucosamine sulfate, gold sodium thiomalate, hydrocodone bitartrate/apap, ibuprofen, risedronate sodium, sulfadiazine, thioguanine, valdecoxib, alefacept, efalizumab and bcl-2 inhibitors.

Non-limiting examples of therapeutic agents for restenosis with which binding proteins of the disclosure can be combined include the following: sirolimus, paclitaxel, everolimus, tacrolimus, Zotarolimus, acetaminophen.

Non-limiting examples of therapeutic agents for sciatica with which binding proteins of the disclosure can be combined include the following: hydrocodone bitartrate/apap, rofecoxib, cyclobenzaprine HCl, methylprednisolone, naproxen, ibuprofen, oxycodone HCl/acetaminophen, celecoxib, valdecoxib, methylprednisolone acetate, prednisone, codeine phosphate/apap, tramadol HCl/acetaminophen, metaxalone, meloxicam, methocarbamol, lidocaine hydrochloride, diclofenac sodium, gabapentin, dexamethasone, carisoprodol, ketorolac tromethamine, indomethacin, acetaminophen, diazepam, nabumetone, oxycodone HCl, tizanidine HCl, diclofenac sodium/misoprostol, propoxyphene napsylate/apap, asa/oxycod/oxycodone ter, ibuprofen/hydrocodone bit, tramadol HCl, etodolac, propoxyphene HCl, amitriptyline HCl, carisoprodol/codeine phos/asa, morphine sulfate, multivitamins, naproxen sodium, orphenadrine citrate, temazepam.

Examples of therapeutic agents for SLE (lupus) with which binding proteins of the disclosure can be combined include the following: NSAIDS, for example, diclofenac, naproxen, ibuprofen, piroxicam, indomethacin; COX2 inhibitors, for example, Celecoxib, rofecoxib, valdecoxib; anti-malarials, for example, hydroxychloroquine; Steroids, for example, prednisone, prednisolone, budenoside, dexamethasone; cytotoxics, for example, azathioprine, cyclophosphamide, mycophenolate mofetil, methotrexate; inhibitors of PDE4 or purine synthesis inhibitor, for example Cellcept. Binding proteins of the disclosure, may also be combined with agents such as sulfasalazine, 5-amino salicylic acid, olsalazine, Imuran and agents which interfere with synthesis, production or action of proinflammatory cytokines such as IL-1, for example, caspase inhibitors like IL-1β converting enzyme inhibitors and IL-1ra. Binding proteins of the disclosure may also be used with T cell signaling inhibitors, for example, tyrosine kinase inhibitors; or molecules that target T cell activation molecules, for example, CTLA-4-IgG or anti-B7 family antibodies, anti-PD-1 family antibodies. Binding proteins of the disclosure, can be combined with IL-11 or anti-cytokine antibodies, for example, fonotolizumab (anti-IFNg antibody), or anti-receptor receptor antibodies, for example, anti-IL-6 receptor antibody and antibodies to B-cell surface molecules. Antibodies of the disclosure or antigen binding portion thereof may also be used with LJP 394 (abetimus), agents that deplete or inactivate B-cells, for example, Rituximab (anti-CD20 antibody), lymphostat-B (anti-BlyS antibody), TNF antagonists, for example, anti-TNF antibodies, Adalimumab (PCT Publication No. WO 97/29131; HUMIRA®), CA2 (REMICADE®), CDP 571, TNFR-Ig constructs, (p75TNFRIgG (ENBREL®) and p55TNFRIgG (LENERCEPT®)) and bl-2 inhibitors, because bcl-2 overexpression in transgenic mice has been demonstrated to cause a lupus like phenotype (see Marquina et al., J. Immunol., 172(11): 7177-7185 (2004)), therefore inhibition is expected to have therapeutic effects.

The pharmaceutical compositions of the disclosure may include a “therapeutically effective amount” or a “prophylactically effective amount” of a binding protein of the disclosure. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the binding protein may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the binding protein to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the binding protein, are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

V. Diagnostics

The disclosure herein also provides diagnostic applications. This is further elucidated below. Binding proteins that bind IL-1α and/or β of the disclosure may be employed in any of a variety of formats to detect IL-1α and/or β in vivo, in vitro, or ex vivo (i.e., in cells or tissues that have been obtained from a living individual, subjected to a procedure, then returned to the individual). DVD-Igs of the disclosure offer the further advantage of being capable of binding to an epitope of IL-1α or β as well as other antigens or epitopes in various diagnostic and detection assay formats. In particular, methods of using IL-1α and β binding proteins as diagnostics are described in U.S. Patent Publication No. 2011/0280800, incorporated by reference, herein, in its entirety.

VI. Kits

A kit for assaying a test sample for the presence, amount or concentration of an analyte (or a fragment thereof) in a test sample is also provided. The kit comprises at least one component for assaying the test sample for IL-1α or β (or fragments thereof) and instructions for assaying the test sample for the analyte (or a fragment thereof). The at least one component for assaying the test sample for the analyte (or a fragment thereof) can include a composition comprising an anti-IL-1α and/or β binding protein, such as a DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof), as described herein and which is optionally immobilized on a solid phase. In particular, kits using IL-1α and β binding proteins and methods of using them are described in U.S. Patent Publication No. 2011/0280800, incorporated by reference, herein, in its entirety.

It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the disclosure described herein are obvious and may be made using suitable equivalents without departing from the scope of the disclosure or the embodiments disclosed herein.

Having now described the binding proteins and methods of making and using them of the disclosure in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting of the disclosure.

EXAMPLES Materials and Methods Materials

1B12.13-SS-X3, human anti-IL-1α/β DVD IgG1/k mut (234,235); PR-1358316 1B12.21-SS-X3, human anti-IL-1α/β DVD IgG1/k mut (234,235); PR-1358317 1B12.34-SS-X3, human anti-IL-1α/β DVD IgG1/k mut (234,235); PR-1358318 1B12.A1-SS-X3, human anti-IL-1α/β DVD IgG1/k mut (234,235); PR-1358319 1B12.A3-SS-X3, human anti-IL-1α/β DVD IgG1/k mut (234,235); PR-1358321 1B12.9, human anti-IL-1β IgG1/k mut (234,235); PR-1317335

Human IL-8 Tissue Culture Kit (K111AN), Meso Scale Discovery MA6000 Methods

MRC-5 Bioassay

MRC-5 cells were grown and cultured in cMEM containing 10% FBS in a 37° C., 5% CO₂ incubator. One day prior to assay, 100 μL of MRC-5 cells were plated well into a 96 well flat bottom plate (Costar#3599) at a density of 1×10⁴ cells per well and incubated overnight at 37° C., 5% CO₂. On the day of the assay, Igs and IL-1α, and IL-1β antigens were prepared at a 4× stock concentration in cMEM. An eight point serial dilution of Igs was prepared at a 4× range of 25,000-0.096 pM in cMEM. Sixty-five μL of diluted Igs was transferred in quadruplicate to a 96 well v-bottom plate (Costar#3894) then 65 μL of a 4× stock of approximately 11 μM of recombinant human IL-1α or recombinant human IL-1β, cyno IL-1β or purified native IL-1β was added to wells containing Igs. For assays using native human IL-1α present in crude supernatants, supernatants were diluted in cMEM to contain approximately 11 pM of native human IL-1α. Antigen control wells received 65 μL of antigen and 65 μL of cMEM media. Cell alone control wells received 130 μL of cMEM media.

Following an hour pre-incubation, 100 μL of IL-1 and Ig complexes was added to the MRC-5 cells. Final well volumes equaled 200 μL and all reagents were at a 1× final concentration. After an overnight incubation (16-24 hour), 150 μL of supernatant was transferred to a 96-well round bottom plate (Costar#3799) and the plates were placed in a −20° C. freezer. The supernatants were tested for human IL-8 levels using a MSD chemiluminescent kit. The neutralization potency of each Ig was determined by calculating percent inhibition relative to the IL-1α or IL-1β control values (in the absence of Ig). IC₅₀ values were calculated using Graph Pad Prism analysis software.

Affinity measurements by Biacore

Goat IgGs specific to human IgG Fc were covalently linked to the carboxy methyl dextran matrix on the CMS biosensor chip via free amine groups using an amine coupling kit and the immobilization wizard option in the Biacore instruments controlling software. Specifically, carboxyl groups of the dextran matrix on the chip were activated with NHS and EDC. Goat anti-human IgG Fc antibodies (25 μg/mL), diluted in 10 mM sodium acetate, pH 4.5, were injected across the activated surface. Once the level of binding response reached the desired value, unreacted groups were deactivated by injection of 1M ethanolamine. For binding of IL-1α & IL-1β to captured Ig, a 1 μg/mL concentration of Ig in HBS-EP+ was injected over the goat anti-human IgG Fc surface at a flow rate of 10 μL/min for 1 minute. The net difference in the baseline signal and the signal after the completion of the Ig injection was taken to represent the amount of bound Ig.

Preformulation Studies

Assays included high concentration drug-like property characterization, accelerated stability at 50 mg/ml (for degradation kinetics), freeze-thaw stability at 50 mg/ml, viscosity at 80 mg/ml, serum stability under crowded conditions, low concentration drug-like property characterization, accelerated stability at 1 mg/ml (for pH optimum determination), freeze-thaw stability at 1 mg/ml (for better resolution of freeze-thaw degradation), biophysical and structural characterization, differential scanning calorimetry (for conformational stability), and FTIR (for secondary structure analysis).

Example 1 Generation of anti-human IL-1β mAb 1B12.9

A new version of h1B12.1 (US 2011/0142761 A1) was generated to allow the characterization of a single amino acid substitution at the N-terminus of the light chain—this new version was called 1B12.9. Humanized monoclonal antibody 1B12.1 (also referred to as h1B12.1) is composed of the heavy chain variable domain 1B12 VH1a and the light chain variable domain VL1a, and was derived from mouse monoclonal 1B12.4H4. 1B12.9 contains the identical heavy chain as 1B12.1, but the light chain variable domain has a single amino acid change at amino acid position 4 in the variable light domain. The valine at position 4 was substituted to a methionine using site-directed mutagenesis, and the resulting light chain plasmid was named h1B12 VL1c.

The V4M mutation was introduced into plasmid pHybE-1B12 VL1a using mutagenic primers (Table 9) and the QuikChange II Site-Directed Mutagenesis kit (Stratagene, cat #200524-5) following the manufacturer's instructions. The pHybE series of expression vectors are described in U.S. Pat. No. 8,187,836 and International PCT Publication No. WO 2009/091912, which are specifically incorporated by reference herein in their entirety. Clones containing the correct sequence were identified by colony sequencing. The DNA of a single clone containing the correct sequence was scaled-up using Qiagen Maxi kit. 1B12.9 was expressed as a human IgG1 mutant (234,235)/k by co-transfection of the heavy chain plasmid (pHybE-1B12 VL1a) and light chain plasmid (pHybeE-1B12 VL1c). Expression of mAb 1B12.9 was performed using small-scale (500 mL) 293 FreeStyle expression system. Light chain, 1B12VH1, and heavy chain, 1B12VL1C, DNA was PEI transfected at a 1:1.5 (heavy chain plasmid to light chain plasmid) ratio. Cell supernatants were purified by protein A chromatography.

TABLE 9 Primers used to construct h1B12 VL1c Primer SEQ ID Name Nucleotide sequence NO:  V4M-F GCGATGCGACACTCAAATGACACAGTCCCCATC 130 V4M-R GATGGGGACTGTGTCATTTGAGTGTCGCATCGC 131

Example 2 Construction of 1B12.1-SS-X3 DVD-Ig

The variable heavy and light chain domains of h1B12.1 were PCR amplified from DNA templates pHybE-h1B12 VH1a and pHybE-h1B12 VL1a, respectively, using primers containing linker sequences. The variable heavy and light chain domains of X₃ were PCR amplified from DNA templates pHybE-X3 VH and pHybE-X3 (1R) VL, respectively, using primers containing linker sequences (Table 10). h1B12.1 VH and X3 VH with the intervening HC-short linker sequence were combined and sub-cloned into pHybE-hCglmut (234,235) at the FspAI and SalI sites via homologous recombination in DH5α chemically competent cells. Likewise, h1B12.1VL and X3 VL with the intervening LC-short linker sequence were sub-cloned into pHybE-hCk at the FspAI and NotI sites. Clones containing the correct sequences were identified by colony sequencing. Expression plasmids were scaled-up using Qiagen Maxi kit.

TABLE 10 Primers used to construct 1B12.1-SS-X3 DVD-Ig SEQ ID Primer Name Nucleotide Sequence NO: 1B12.1-X3- ACCGTGTCCAGTGCCTCAACAAAAGGGCCT 112 VH-F CAGGTGCAGCTGGTGGAAAG 1B12.1-X3- CAGCTGCACCTGAGGCCCTTTTGTTGAGGCA 113 VH-R CTGGACACGGTCACAAGTG 1B12.1-X3- CTTGAGATCAAGCGTACGGTGGCTGCACCA 114 VL-F GATATTCAGATGACCCAGAG 1B12.1-X3- GGTCATCTGAATATCTGGTGCAGCCACCGTA 115 VL-R CGCTTGATCTCAAGCTTCG 1B12-VH1a-F CTGGCTTTTTCTTGTCGCGATTTTAAAAGGT 116 GTCCAGTGCGAGGTACAACTTCAAGAATC 1B12-VL1a-F GCTGGGCCTGCTGCTGCTGTGGTTCCCCGGC 117 TCGCGATGCGACACTCAAGTGACACAGTC VHRO hCg1 AGGGTGCCAGGGGGAAGACCGATGGGCCCT 118 5′ Rev TGGTCGACGC 7416 CCTTTATTAGCCAGAGGTCGAGGTC 119

For DVD-Ig protein expression, the 1B12.1-SS-X3 DVD-Ig heavy chain pHybE expression vector and 1B12.1-SS-X3 DVD-Ig light chain pHybE expression vector were co-transfected into 293-6E HEK cells in a small-scale expression (500 mL) 293 FreeStyle expression system. The expression level of DVD-Ig was measured by human Ig ELISA and determined to be approximately 45 ug/ml.

Example 3 Humanized IL-1β h1B12.1 affinity maturation design

Sequence alignment shows that the IL-1β antibody h1B12.1 shares the highest identity to human germlines VH4-59/JH4 and IGKV1-39/Jk2. To improve the affinity of h1B12.1 to IL-1β, hypermutated CDR residues were identified from other human antibody sequences in the IgBLAST database that also shared high identity to germlines VH34-59 and IGKV1-39. The corresponding h1B12.1 CDR residues were then subjected to limited mutagenesis by PCR with primers having low degeneracy at these positions to create three antibody libraries in the scFv format suitable for yeast surface display. The first library (H1+H2) contained mutations in HCDR1 and HCDR2. The second library (H3) contained mutations in HCDR3 and the third library (LC) contained mutations in all LCDRs. To further increase the identity of h1B12.1 to the human germline framework sequences, a binary degeneracy at certain positions were introduced into the libraries. The library designs are indicated below:

CDRs H1 and H2: Introduce limited mutagenesis at nine amino acid positions (30, 31, 32, 51, 52, 53, 54, 56, and 58) and in VH.1a and germline (F27G, L48I, K71V, N73T) CDR H3: Introduce limited mutagenesis at eleven amino acid positions (95-100, 100a-100e) and toggle between VH.1a and germline: K94R CDRs L1, L2 and L3: Introduce limited mutagenesis at eleven amino acid positions (30, 31, 32, 50, 53, 55, 56, and 91-94) at 79-7-7-7 ratio and in VL.1d and germline (T21,124Q, T25A, T27Q, M33L, S49Y, G51A, N52S, S64G, L89Q) These h1B12.1 libraries were transformed into yeast cells and displayed on cell surfaces to be selected against a low concentration of biotinylated human and/or cynoIL-1β by magnetic then fluorescence activated cell sorting.

Example 4 h1B12 scFv Expression and Binding Analysis on the Surface of Yeast

h1B12.1 was expressed as scFv on the surface of yeast using the pYD1 yeast display vector (Invitrogen). The scFv-expressing yeast were exposed to different biotinylated antigen concentrations under different time and temperature conditions. ScFv expression was monitored by tag-specific antibodies. Fluorochrome labeled donkey anti-mouse (PerCP), goat (PE) or rabbit (DyLight488) antibodies from Jackson Immunoresearch were used as detection reagents. Antigen binding was monitored by APC conjugated streptavidin or Dylight633 conjugated neutravidin. All samples were analyzed by flow cytometry using a FACS Canto II cytometer and FACS Diva software version 4.3. Tag specific antibodies are described in Table 11.

The best Ag binding conditions for library selections were first identified by scouting experiments. We looked for conditions that best discriminate library populations with different binding affinities towards IL-1β. These conditions allowed detection of bound Ag and library-specific tags to normalize the antigen-binding signal for expression. The next step was to sort and collect library for further analysis. The cells were run in a FACSAria II cell sorter and the clones to select were gated according to their binding and expression. In each round of selection (see Table 12 for details) clones with the best affinity and expression were collected.

Selection for improved on-rate, off-rate, or both were carried out and antibody protein sequences of affinity-modulated h1B 12.1 variants (see below) were recovered from yeast cells for converting back to IgG format for further characterization.

TABLE 11 Commercially available anti-peptide tagged antibodies used to monitor ScFv antibody expression on yeast Tag Ab Source Clone Company Cat # S Mouse SBSTAGa Abcam ab24838 S Rabbit Polyclonal S tag antibody ab18588 [SBSTAGa] AcV5 Mouse AcV5 Abcam. Rabbit S tag ab49581 antibody E2 Mouse 5E11 Abcam. AcV5 tag ab977 antibody [AcV5] E Rabbit Polyclonal T7 tag ® antibody ab3397 [T7 Tag] E Goat Polyclonal Abcam ab95868 E Chicken Polyclonal KT3 tag antibody [KT3] ab18695 StrepII Mouse Strep-tag Abcam. E tag antibody MCA2489 StrepII Rabbit Polyclonal Abcam. E tag antibody A00626 HA Mouse HA-7 Sigma H9658 HA Goat Polyclonal Abcam ab9134 HA Rat (IgG1) 3F10 Roche 11 867 423 c-myc Mouse 9E10 Sigma M4439 c-myc Rabbit Polyclonal Sigma C3956 Flag Mouse M2 Sigma F3165 Flag Rabbit Polyclonal Sigma F7425 HSV Rabbit Polyclonal Sigma H6030

TABLE 12 Commercially available secondary reagents used to monitor scFv antibody expression and binding on the surface of yeast Secondary reagent Fluorocrome Company Cat # F(ab′)2 Frag. Donkey PerCp Jackson Anti-Rat IgG (H + L) ImmunoResearch F(ab′)2 Frag. Donkey R-PE Jackson Anti-Goat IgG (H + L) ImmunoResearch F(ab′)2 Frag. Donkey DyLight-488 Jackson Anti-Rabbit IgG (H + L) ImmunoResearch F(ab′)2 Frag. Goat R-PE Jackson Anti-Rabbit IgG (H + L) ImmunoResearch F(ab′)2 Frag. Goat Anti-Rabbit Alexafluor Invitrogen IgG (H + L) Chicken anti mouse IgG (H + L) PerCP Jackson ImmunoResearch F(ab′)2 Frag Donkey Anti- Alexafluor ThermoScientific Mouse IgG(H + L) Streptavidin APC Streptavidin PE Neutravidin PE Neutravidin DyLight-633

Example 5 Generation of affinity-matured h1B12.1 IgG1/k (mut (234,235) mAbs

Five h1B12 affinity matured antibodies named h1b12-AM1-5 were produced in HEK-293-6E cells. All mAbs were generated as human IgG1/k mut (234,235) constructs. They were produced via small-scale transfections with a 200 mL transfection volume. The supernatants were harvested on day 6 post transfection. The titer of an aliquot of the supernatant was determined by human Ig ELISA in the assay lab. MabSelect SuRe resin (GE Healthcare) was used for affinity purification. Following elution, the antibodies were dialyzed against 15 mM histidine pH 6 overnight. The solutions were sterile filtered and analyzed by mass spectrometry and SEC for identity and purity confirmation.

Example 6 Indirect IL-1β Binding ELISA

An indirect ELISA was performed to assess the relative affinity of the affinity matured h1B12.1 mAbs to a human chimeric version of the murine 1B12 mAb. An ELISA plate was coated with goat anti-human IgG (Jackson Immunoresearch cat#109-005-098) at 0.5 ug/mL overnight. The plate was blocked with 5% milk for 1 hour and washed three times with TPBS. Affinity-matured h1B12.1 antibodies and human IgG control (Sigma cat#12511) were incubated on the plate for 30 min at 0.2 μg/mL and then the plates were washed three times. A titration of biotinylated human IL-1β or biotinylated cyno IL-1β was added to the plate, starting at 10 nM and decreasing in concentration following a series of 1:3 dilutions. Binding of biotinylated IL-1β was detected with a 1:10000 dilution of peroxidase-conjugated streptavidin (Jackson Immunoresearch cat#016-030-084) and TMB substrate (Invitrogen). The data were fit in Graphpad prism to determine the EC₅₀ values.

Example 7 Expression of 5 New DVD-Igs from Affinity-Matured h1B12.1 Variable Domains

Five DVD-Ig molecules were generated with the affinity-matured h1B12 variable domains and the X3 variable domain with the light chain short linker sequence TVAAP (SEQ ID NO:42) and the heavy chain short linker sequence ASTKGP (SEQ ID NO:43). These are referred to as DVD1-h1B12.13-SS-X3 (PR-1358316), DVD2-h1B12.21-SS-X3 (PR-1358317), DVD3-h1B12.34-SS-X3 (PR-1358318), DVD4-h1B12.A1-SS-X3 (PR-1358319), DVD5-h1B12.A3-SS-X3 (PR-1358321). The affinity-matured heavy chain and light chain 1B12 variable regions were PCR'd out using overlapping primers from the monoclonal heavy chain and light chain constructs (Tables 13 and 14). The X3 VH and VL (including the short-short linker with overlapping primers) were also PCR'd out using the 1B12.1.5S.X3 VH and VL DVD constructs as templates. The two VH and VL with the linker were then combined with overlapping PCR. Using homologous recombination these constructs were cloned into the linearized vector pHybE hCG1 mut (234,235) digested with FSPA1 and Sail for heavy chain and pHybE hCk digested with FSPAI and BSWiI for light chain followed by transformation into DHSa chemically competent cells. Correct clones identified by colony sequencing were scaled up. The Fc region of these five DVD-Ig molecules is IgG1 mutant 234,235. Table 15 lists the heavy chain and light chain constructs used to express each IL-1α/β back up DVD-Ig. For protein expression, a heavy chain pHybE expression vector and light chain pHybE expression vector were co-transfected into 293 cells, followed by purification using Protein A chromatography. In a small-scale expression (500 mL) in 293 freestyle expression system, the expression levels of DVD-Ig molecules were determined by human Ig ELISA.

TABLE 13 Heavy chain primer sequences Heavy chain primer name Nucleotide sequence SEQ NO: VH1B12.13. TGGCTTTTTCTTGTCGCGATTTTAAAAGG 120 ss.X3.F1 TGTCCAGTGCGAGGTACAACTTCAAGAA TCTGGTCCAGGG VH1B12.13. GCTTTCCACCAGCTGCACCTGCGGTCCTT 121 ss.X3.R1 TAGTGCTAGCACTGGACACGGTCACAAG TGTGCCTTGGCC VH1B12.13. ACACTTGTGACCGTGTCCAGTGCTAGCA 122 ss.X3.F2 CTAAAGGACCGCAGGTGCAGCTGGTGGA AAGCGGCGGCGGCGTG VH1B12.13. GGGTGCCAGGGGGAAGACCGATGGGCCC 123 ss.X3.R2 TTGGTCGACGCGCTGCTAAAGGTCACCA GGGTGCCCTGGCCCCA

TABLE 14 Light chain primer sequences Light chain primer name Nucleotide sequence SEQ NO: VL1B12.13. CTGGGCCTGCTGCTGCTGTGGTTCCCCGG 124 ss.X3.F1 CTCGCGATGCGACATTCAAATGACACAG TCCCCATCATCTTTG VL1B12.13. GCTCTGGGTCATCTGAATATCAGGAGCT 125 ss.X3.R1 GCGACGGTACGCTTGATCTCAAGCTTCGT ACCTTGCCCGAAAGT VL1B12.13. CAAGGTACGAAGCTTGAGATCAAGCGTA 126 ss.X3.F2 CCGTCGCAGCTCCTGATATTCAGATGACC CAGAGCCCGAGCAGCGTGAGC VL1B12.6. GAAGATGAAGACAGATGGTGCAGCCACC 127 13.ss.X3.R2 GTGCGTTTATGTTCCACTTTGGTGCCGCC GCCAAAGCTCAG

TABLE 15 pHybE Expression plasmids to generate IL-1 α/β back up DVD-Igs Heavy chain Light chain DVD-Ig plasmid plasmid h1B12.13.SS.X3 pHybE-hCg1, mut(234,235), pHybE hCk h1B12.13-SS-X3 VH h1B12.13.SS.X3 VL h1B12.21.SS.X3 pHybE-hCg1, mut(234,235) pHybE hCk h1B12.21-SS-X3 VH h1B12.21.SS.X3 VL h1B12.34.SS.X3 pHybE-hCg1, mut(234,235) pHybE hCk h1B12.34-SS-X3 VH h1B12.34.SS.X3 VL h1B12.A1.SS.X3 pHybE-hCg1, mut(234,235) pHybE hCk h1B12.A1-SS-X3 VH h1B12.A1.SS.X3 VL h1B12.A3.SS.X3 pHybE-hCg1, mut(234,235) pHybE hCk h1B12.A3-SS-X3 VH h1B12.A3.SS.X3 VL

Example 8 Characterization of 1B12.1-SS-X3 DVD-Ig and anti-human IL-1β mAb 1B12.9

The 1B12.1-SS-X3 DVD-Ig IgG1/k mut (234,235) DVD contains the light chain short linker sequence TVAAP (SEQ ID NO: 42) and the heavy chain short linker sequence ASTKGP (SEQ ID NO: 43). Anti-human IL-1β mAb 1B12.9 was generated as described above. Characterization data for both molecules is shown in Tables 16, 17, 18a, and 18b.

TABLE 16 Expression data on Igs 1B12.1-SS-X3 and 1B12.9 purified from 500 mL HEK293 cell supernatant Concentration of Purified mAb/DVD Total Yield mAb/DVD Name (mg/mL) (mg) 1B12.9 2.3 33 1B12.1-SS-X3 2.1 10.5

TABLE 17 Characterization of 1B12.1-SS-X3 DVD-Ig and 1B12.9 by SEC Ig Name % Monomer 1B12.9 92 1B12.1-SS-X3 87

TABLE 18a Characterization of 1B12.1-SS-X3 DVD-Ig by Biacore Human Human Cyno Cyno IL-1β IL-1α IL-1β IL-1α K_(on) (1/Ms) >1.0E+06 2.1E+05 7.3E+06 7.4E+04 K_(off) (1/s)  8.2E−04 2.4E−05 8.0E−04 2.6E−04 K_(D) <8.2E−10 1.2E−10 1.1E−10 3.5E−09

TABLE 18b Characterization of 1B12.1-SS-X3 DVD-Ig by Biacore 1B12.9 hu IgG1/k mut (234,235), PR-1317335, Lot# 1808846 Antigens k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) Human IL-1β Expt 1 5.5E+06 7.6E−04 1.4E−10 PR-1317335 Expt 2 5.8E+06 7.7E−04 1.3E−10 lot 1808846 Average 5.7E+06 7.7E−04 1.4E−10 Cyno IL-1β Expt 1 2.0E+06 9.8E−04 4.9E−10 A-1198637 Expt 2 1.4E+06 8.5E−04 6.0E−10 lot 1667136 Average 1.7E+06 9.2E−04 5.5E−10

Example 9 Potency of IgGs

The neutralization potency of 1B12.9 and 1B12.1-SS-X3 DVD-Ig on human and cynomolgus IL-1β and native human IL-1β was assessed by the MRC-5 cell-based bioassay. 1B12.1 was used as a positive control comparator in the assay. In addition, the potency of 1B12.1-SS-X3 DVD-Ig on human and cynomolgus IL-1α and native human IL-1α was also assessed (Table 19).

TABLE 19 Neutralization potency determined by MRC-5 bioassay. Potency IC50 (pM) Human Cynomolgus Native mAb/DVD-Ig IL-1a IL-1b IL-1a IL-1b IL-1a IL-1b 1B12.9 691 352 214 PR-1317335 1B12.1 913 410 329 PR-1262922 1B12.1.SS.X3 27 26 2508 461 79 156 PR-1308565

Example 10 h1B12.1 Libraries Binding and Selection Using Human and Cyno IL-1β

The h1B12.1 scFv clone was able to bind human and cyno IL-1β with affinities in the 10 to 30 nM range. Both the on-rates and off-rates were targeted for improvement following the rounds of selection described in Table 20.

TABLE 20 H1 + H2, H3 and LC Libraries selection conditions Off rate competition Selection w/ unlabeled IL- round Library Ag [Ag] Time Temp. 1β at 37° C. M1 All  30 nM huIL-1b 30 min  37° C. M2 h1B12 H1 + H2   1 nM cynoIL-1b 3 min Ice h1B12 H3 M2 h1B12 LC   1 nM cynoIL-1b 5 min Ice M2S1 h1B12 H1 + H2 0.3 nM huIL-1b 3 min Ice h1B12 H3 M2S1 h1B12 LC 0.3 nM huIL-1b 5 min Ice M2S2-2 h1B12 H1 + H2 0.3 nM huIL-1b 2 min Ice M2S2-2 h1B12 H3 0.1 nM huIL-1b 2 min Ice h1B12 LC M2S3-2 h1B12 LC   1 nM huIL-1b 2 min ice

H1+H2, H3 and LC libraries displayed at least 10 fold improved binding to IL-1β compared to parental h1B12 scFv. However, improvement of 100 fold was highly desirable. To achieve affinity improvements >10-fold, a new h1B12 scFv library recombining the improved sequences from all 3 selected libraries was then generated and again selected for clones with improved human and cyno IL-1β binding following the rounds of selection on Table 21.

TABLE 21 Recombined h1B12 (rHC + LC) library selection conditions Off rate competition Selection round Library Ag [Ag] Time Temp. w/ unlabeled IL-1β at 37° C. M1 h1B12 rHC + LC 1 nM huIL-1b 2 min Ice 2 hour M1S1 h1B12 rHC + LC 0.04 nM huIL-1b   1 min Ice M1S2 h1B12 rHC + LC 1 nM huIL-1b 2 min ice 6 hour M1S3-2 h1B12 rHC + LC 1 nM huIL-1b 2 min ice 24 hour  M1S4 h1B12 rHC + LC 0.03 nM huIL-1b   30 min  RT

Example 11 Affinity of the h1B12.1 rHC+LC Library is More than 150 Fold Improved Compared to Parental h1B12.1 scFv

A binding analysis comparison of the h1B12.1 scFv and the final M1S3-2 library output was generated in order to show the relative improvement in affinity of the library output. H1B12 and rHC+LC M1S3-2 were exposed to different concentrations of human and cyno IL-1β. The analysis shows a comparable binding to human or cyno IL-1β and at least 150 fold improvement in the binding of M1S3-2 output over h1B12 parental scFv (FIG. 1).

The sequences of 12 variable heavy chain domains (FIG. 2) and 12 variable light chain domains (FIG. 3) were aligned with h1B12.1 to show the sequence changes resulting in affinity improvements. Within these 12 sequences, there are 2 duplications in each of the heavy chain and light chain sequences, so a final total of 10 different clones were identified with improved binding to human and cyno IL-1β were identified from M1S3-2 output (off-rate selection) and M1S4 selection (equilibrium binding).

Example 12 Protein Sequence Liability Screening of h1B12.1 and Affinity Matured Variants

h1B12.1 affinity matured variants were screened for potential liability sequence motifs, including deamidation, oxidation, isomerization, cleavage, hydrolysis, and N-linked glycosylation. IgG proteins should be characterized by the appropriate assays with sufficient sensitivity to confirm the presence or absence of each of the potential post-translational modifications.

Further analysis of protein sequence diversity and sequence prevalence in the final outputs were used to select five lead h1B12.1 improved variants which received the name of h1B12-AM1 to AM5 (Table 22a).

TABLE 22a h1B12.1 affinity matured clones; nomenclature from scFv to IgG Affinity Affinity matured full matured length IgG scFv output HC plasmid LC plasmid name 13 h1B12 pHybE-hCg1mut-h1B12-13 pHybE-hCk-h1B12-13 h1B12-AM1  21* rHC + LC pHybE-hCg1mut-h1B12-21 pHybE-hCk-h1B12-21 h1B12-AM2 34 M1S3-2 pHybE-hCg1mut-h1B12-34 pHybE-hCk-h1B12-34 h1B12-AM3 A1 M1S4 pHybE-hCg1mut-h1B12-A1 pHybE-hCk-h1B12-A1 h1B12-AM4 A3 pHybE-hCg1mut-h1B12-A3 pHybE-hCk-h1B12-A3 h1B12-AM5 *Clone 21 is found in both outputs

Example 13 Generation of h1B12.1 Affinity-Matured IgGs

Following the identification of 5 affinity-matured versions of 1B12.1, each of the 5 variable domain sequences were expressed as a monoclonal IgG1/k mut (234,235) antibody. Each antibody was tested by ELISA to determine if the affinity had been improved to both human and cyno IL-1β, and the chimeric mouse/human 1B12 IgG1/k mut (234,235) was used as a comparator in these assays (Table 22b). All five affinity matured mAbs had EC₅₀ values <0.05 nM against human and cyno IL-1β (values are comparable to one another) and have improved affinity compared to chimeric 1B12 (Table 22c).

TABLE 22b Information and expression data and on affinity-matured h1B12.1 mAbs mg mAb Titer purified from 200 mL SEC mAb Identifier Lot (ug/mL) supernatant (% monomer) h1B12-AM-1 PR-1354589 1851887 23 5.0 98.5 h1B12-AM-2 PR-1354591 1851889 128 23.2 99 h1B12-AM-3 PR-1354592 1851890 72 14.7 99 h1B12-AM-4 PR-1354594 1851892 67 9.5 99.5 h1B12-AM-5 PR-1354603 1851902 23 4.1 98 Chimeric 1B12 A-867628 1274704 N/A N/A N/A (mouse/human)

TABLE 22c EC50 values generated following analysis of affinity- matured h1B12.1 mAbs by direct-antigen binding ELISA ELISA EC50 (nM) mAb Human IL-1β Cyno IL-1β Chimeric 1B12 0.107 0.087 (mouse/human) h1B12-AM-1 0.031 0.026 h1B12-AM-2 0.026 0.034 h1B12-AM-3 0.033 0.032 h1B12-AM-4 0.031 0.025 h1B12-AM-5 0.028 0.031

Example 14 Characterization of DVD-Igs Containing Affinity-Matured 1B12 Variable Domains

Five DVD-Igs containing affinity-matured 1B12.1 IL-1β-binding domains paired with the IL-1α-binding domain X3 were generated as discussed in Materials and Methods. The total yields after purification of 500 mL culture supernatants and characterization data are shown in Table 23.

TABLE 23 Purification of DVD-Ig molecules in 293 freestyle system Concentration of Total yield Purified DVD from 500 mL SEC (% DVD (mg/mL) (mg) monomer) h1B12.13-SS-X3 0.29 1.39 98 h1B12.21-SS-X3 0.56 2.52 89 h1B12.34-SS-X3 0.55 3.3 94 h1B12.A1-SS-X3 0.32 2.08 95 h1B12.A3-SS-X3 0.19 0.95 96

Example 15 Characterization of 5 DVD-Igs Containing Affinity-Matured h1B12.1 Domains by MRC-5 Bioassay and Biacore

The potency of the 5 DVD-Igs containing affinity matured h1B12.1 domains to recombinant and native IL-1 antigens was determined using the MRC-5 bioassay as shown in Table 24. The binding kinetics of the DVD-Igs to recombinant human and cynomolgus IL-1α and IL-1β were determined using surface plasmon resonance. The affinity determinations are shown in Table 25. In some cases, the off-rate could not be determined as they reached the limit of detection of the instrument. For these samples (indicated with a star), the overall affinity was calculated assuming the off-rate to be 1×10⁻⁶.

TABLE 24 Potency of DVD-Igs containing affinity-matured 1B12.1 variable domains in MRC-5 bioassay Potency (IC50) pM Human Cynomolgus Native DVD-Ig IL-1α IL-1β IL-1α IL-1β IL-1α IL-1β h1B12.13-SS-X3 45.4 55.9 2439 3.62 24.4 1.64 PR-1358316 h1B12.21-SS-X3 42.8 48.6 1642 1.86 72.0 1.91 PR-1358317 h1B12.34-SS-X3 41.3 42.2 1895 2.13 50.4 0.998 PR-1358318 h1B12.A1-SS-X3 38.6 58.6 2584 4.02 50.9 2.46 PR-1358319 h1B12.A3-SS-X3 44.3 51.2 1921 6.56 57.6 4.27 PR-1358321

TABLE 25 Affinity of DVD-Igs containing affinity-matured 1B12.1 variable domains by BIAcore Sample Ligand k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) h1B12.13-SS-X3 Hu IL-1alpha 1.4E+05 2.9E−06 2.1E−11 h1B12.21-SS-X3 Hu IL-1alpha 1.4E+05 1.6E−05 1.2E−10 h1B12.34-SS-X3 Hu IL-1alpha 1.4E+05 4.2E−06 3.0E−11 h1B12.A1-SS-X3 Hu IL-1alpha 1.8E+05 3.6E−05 2.0E−10 h1B12.A3-SS-X3 Hu IL-1alpha 1.5E+05 1.6E−06 1.0E−11 h1B12.13-SS-X3 Hu IL-1beta 2.3E+07 5.1E−06 2.2E−13 h1B12.21-SS-X3 Hu IL-1beta 2.5E+07 5.1E−06 2.0E−13 h1B12.34-SS-X3 Hu IL-1beta 3.9E+07 *<1E−06 *<2.6E−14 h1B12.A1-SS-X3 Hu IL-1beta 1.1E+07 1.0E−05 9.8E−13 h1B12.A3-SS-X3 Hu IL-1beta 2.6E+07 1.3E−05 5.0E−13 h1B12.13-SS-X3 Cyno IL-1alpha 1.0E+05 2.3E−04 2.3E−09 h1B12.21-SS-X3 Cyno IL-1alpha 1.0E+05 2.1E−04 2.0E−09 h1B12.34-SS-X3 Cyno IL-1alpha 1.0E+05 2.4E−04 2.3E−09 h1B12.A1-SS-X3 Cyno IL-1alpha 1.3E+05 3.5E−04 2.6E−09 h1B12.A3-SS-X3 Cyno IL-1alpha 1.1E+05 2.7E−04 2.4E−09 h1B12.13-SS-X3 Cyno IL-1beta 1.2E+07 1.7E−05 1.5E−12 h1B12.21-SS-X3 Cyno IL-1beta 1.1E+07 1.8E−05 1.6E−12 h1B12.34-SS-X3 Cyno IL-1beta 1.8E+07 1.2E−05 6.7E−13 h1B12.A1-SS-X3 Cyno IL-1beta 5.3E+06 9.6E−06 1.8E−12 h1B12.A3-SS-X3 Cyno IL-1beta 1.3E+07 1.3E−05 1.0E−12

Example 16 Preformulation Studies

Two DVD-Igs (h1B12.13-SS-X3 and h1B12.A3-SS-X3) were scaled up to approximately 30 mg for a preformulation analysis and results are shown in Table 26. The assays were performed in 15 mM histidine pH 6 with a DVD-Ig concentration of 50 mg/mL. Table 26 indicates concentrating h1B12.13-SS-X3 was faster than concentrating h1B12.A3-SS-X3 even though h1B12.13-SS-X3 had much higher viscosity. Additional preliminary studies (e.g. formulation in water) suggested higher ionic strength may reduce viscosity to acceptable levels (<10 cPs). All other properties (storage stability, freeze-thaw stability, serum stability) were within typical monoclonal antibody ranges.

TABLE 26 Preformulation data on two DVD-Igs High Conc. High Conc. Freeze Accelerated Thaw Stability High Conc. 5° C. Stability Δ (% HMW) post 4 Viscosity Stability Δ (% M) loss @ freeze thaw (at 80 mg/mL) Serum Stability Δ (% M) loss T21 d, 40° C. cycles cPs @ 20° C. % HMW/day @ 5° C. h1B12.13-SS-X3 6.1% −1.8% 160 0.24% 0.01% @ 23 days h1B12.A3-SS-X3 7.0% −0.1% 16 0.12% 0.01% @ 23 days

The two DVD-Igs (h1B12.13-SS-X3, PR-1358316 and h1B12.A3-SS-X3, PR-1358321) were also characterized in a single-dose rodent PK study and results are shown in FIG. 4 and FIG. 5.

Example 17 Pharmacokinetic Studies

PR-1358321 and PR-1358316 were administered to CD-1 mice and Sprague-Dawley rats by slow intravenous bolus dose injection at a 4 or 5 mg/kg (rat and mice respectively) dose. Blood samples were collected from each mouse at 1, 24 and 96 hours and 7, 10, 14 and 21 days post dose. Blood samples were collected from each rat at 0.25, 4, and 24 hours and 2, 3, 7, 10, 14, 21 and 28 days post dose. All samples were stored at −80° C. until analysis. Serum samples were analyzed using a IL-1α/β hybrid MSD assay employing a biotinylated rhIL-1 beta for capture and Sulfo-Tag labeled human IL-1alpha for detection. The assay was carried out in 1% final serum concentration. The lower limit of quantitation (LLOQ) was 0.15 μg/mL for both mouse and rat studies.

Standard curve fitting and data evaluation was performed using XLfit4 software with a four-parameter logistic fit. Plates passed when at least ⅔ of the QC's were within 30% of the expected values. Pharmacokinetic parameters for each animal were calculated using WinNonlin software Version 5.0.1 (Pharsight Corporation, Mountain View, Calif.) by non-compartmental analysis using linear trapezoidal fit (NCA Models #201 for IV dosing). For calculations in WinNonlin, the time of dosing was defined as Day 0 Time 0 hr.

Both DVD-Igs demonstrated low clearance (0.16 and 0.19 mL/h/kg) with small volumes of distribution (109 and 94 mL/kg) and long half-lives (19 and 14 days) in CD-1 mouse. In Sprague-Dawley rats, both DVDs also displayed low CL (0.21 and 0.24 mL/h/kg) and small volume of distribution (78 and 71 mL/kg), with PR-1358316 and PR-1358321 displaying a half-life respectively (9 and 12 days). 

1. A binding protein that specifically binds to both IL-1α and IL-1β, said binding protein comprising an IL-1β antigen binding domain comprising six CDRs: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, as defined below: CDR-H1: X1-X2-G-V-S (SEQ ID NO:146), wherein; X1 is D, E, H, V or Q and X2 is Y, F, D, N, H or S; CDR-H2: X3-I-X4-X5-X6-G-X7-T-X8-Y-N-S-P-L-K-S (SEQ ID NO:147), wherein: X3 is L, V, R, G, F, W, M or I; X4 is W, G, L, Y, S, R, E or A; X5 is G, V, R, W, L, D or S; X6 is G, R, V, W, S, L, I, E or D; X7 is D, G, E, V, S, A, K, D, Y or Q; and X8 is Y, F, S, D, V, T or H; CDR-H3: X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19M-D-Y (SEQ ID NO:148), wherein; X9 is Q, D, K, P, H, A, Y, V or T; X10 is R, T, M, S, I, W, P, N or K; X11 is T, R, N, Y, W, V, M or K; X12 is L, M, R, I, G, W, Q, H, F or A; X13 is W, S, G, R, V, L, K, A, Y or T; X14 is G, A, R, V, T, P, N, E or D; X15 is Y, F, N, D, H or S; X16 is D, Q, H, A, V, L or K; X17 is L, I, F, S, M or G; X18 is Y, N, D, V or D; and X19 is G, A, R, D or W; CDR-L1: X20-X21-S-X22-D-I-X23-X24-X25-M-N (SEQ ID NO:149), wherein: X20 is I or Q; X21 is T or A; X22 is T or Q; X23 is D, P, Y, N, Q, or L; X24 is V, P, A or F; and X25 is D, A, P, N, Y or V; CDR-L2 have the sequence X26-X27-X28-X29-L-X₃₀-X31 (SEQ ID NO:150), wherein: X26 is S or Y; X27 is Q, H, P, K or R; X28 is G or A; X29 is N or S; X30 is T, S, I, M or A; and X31 is P, L, S or T; and CDR-L3: X32-Q-X33-X34-X35-X36-X37-L-T (SEQ ID NO:151), wherein; X32 is L or Q; X33 is S, W, R, K, G or V; X34 is D, G, K or V; X35 is N, E, R, M or G; X36 is L, G, D or F; and X37 is P or A.
 2. The binding protein according to claim 1, wherein at least one CDR comprises an amino acid sequence selected from the group consisting of: residues 31-35 of SEQ ID NOs: 30, 31, 32, 33 and 34; residues 50-65 of SEQ ID NOs: 30, 31, 32, 33 and 34; residues 98-111 of SEQ ID NOs: 30, 31, 32, 33 and 34; residues 24-34 of SEQ ID NOs: 36, 37, 38, 39 and 40; residues 50-56 of SEQ ID NOs: 36, 37, 38, 39 and 40; and residues 89-97 of SEQ ID NOs: 36, 37, 38, 39 and
 40. 3. The binding protein according to claim 2, wherein at least two CDRs comprise an amino acid sequence selected from the group consisting of: residues 31-35 of SEQ ID NOs:30, 31, 32, 33 and 34; residues 50-65 of SEQ ID NOs: 30, 31, 32, 33 and 34; residues 98-111 of SEQ ID NOs: 30, 31, 32, 33 and 34; residues 24-34 of SEQ ID NOs: 36, 37, 38, 39 and 40; residues 50-56 of SEQ ID NOs: 36, 37, 38, 39 and 40; and residues 89-97 of SEQ ID NOs: 36, 37, 38, 39 and
 40. 4. The binding protein according to claim 2, wherein at least three CDRs comprise an amino acid sequence selected from the group consisting of: residues 31-35 of SEQ ID NOs:30, 31, 32, 33 and 34; residues 50-65 of SEQ ID NOs: 30, 31, 32, 33 and 34; residues 98-111 of SEQ ID NOs: 30, 31, 32, 33 and 34; residues 24-34 of SEQ ID NOs: 36, 37, 38, 39 and 40; residues 50-56 of SEQ ID NOs: 36, 37, 38, 39 and 40; and residues 89-97 of SEQ ID NOs: 36, 37, 38, 39 and
 40. 5. The binding protein according to claim 2, comprising six CDRs, wherein between four and six CDRs comprise an amino acid sequence selected from the group consisting of: residues 31-35 of SEQ ID NOs:30, 31, 32, 33 and 34; residues 50-65 of SEQ ID NOs: 30, 31, 32, 33 and 34; residues 98-111 of SEQ ID NOs: 30, 31, 32, 33 and 34; residues 24-34 of SEQ ID NOs: 36, 37, 38, 39 and 40; residues 50-56 of SEQ ID NOs: 36, 37, 38, 39 and 40; and residues 89-97 of SEQ ID NOs: 36, 37, 38, 39 and
 40. 6. The binding protein according to claim 1, comprising six CDRs: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 CDR-H1: X₁-X₂-G-V-S (SEQ ID NO:1), wherein; X₁ is D or E and X₂ is Y or F; CDR-H2: L-I-W-G-X₃ G-D-T-Y-Y-N-S-P-L-K-S (SEQ ID NO:2), wherein: X₃ is S or G; CDR-H3: Q-X₄-N-X₅-W-X₆-Y-D-L-Y-X₇-M-D-Y (SEQ ID NO:3), wherein; X₄ is T or R; X₅ is L or I; X₆ is A or G and X₇ is S or G; CDR-L1: Q-X₈-S-X₉-D-I-D-X₁₀-D-X₁₁-N (SEQ ID NO:4), wherein; X₈ is A or T; X₉ is Q or T; X₁₀ is D or M; and X₁₁ is L or M; CDR-L2: X₁₂-X₁₃-X₁₄-X₁₅-L-X₁₆-P (SEQ ID NO: 5), wherein; X₁₂ is L or Q; X₁₃ is A or G; X₁₄ is S or M; X₁₅ is I, K or T; and X₁₆ is R, W or P; and CDR-L3: L-Q-X₁₇-D-X₁₈-X₁₉-P-L-T (SEQ ID NO: 6), wherein; X₁₇ is S or T; X₁₈ is S, W or R; and X₁₉ is F or L.
 7. The binding protein according to claim 1, wherein the binding protein is capable of neutralizing IL-1.
 8. The binding protein according claim 1, wherein the binding protein has an IC50 of between 30 and 60 p M using the MRC-5 bioassay.
 9. The binding protein according claim 1, further comprising an IL-1α antigen binding domain comprising a CDR-H1 comprising an amino acid sequence of SEQ ID NO:132, a CDR-H2 comprising an amino acid sequence of SEQ ID NO:133, and a CDR-H3 comprising an amino acid sequence of SEQ ID NO:134, a CDR-L1 comprising an amino acid sequence of SEQ ID NO:135, a CDR-L2 comprising an amino acid sequence of SEQ ID NO:136, and a CDR-L3 comprising an amino acid sequence of SEQ ID NO:137.
 10. The binding protein of claim 1, wherein the binding protein is cleared from a CD-1 mouse subject at between 0.16 and 0.19 mL/h/kg when administered at 4-5 mg/kg.
 11. The binding protein of claim 1, wherein the volume of distribution of the binding protein is between 109 and 94 mL/kg when the binding protein is administered to a CD-1 mouse subject at 4-5 mg/kg.
 12. The binding protein of claim 1, wherein the half-life of the binding protein is between 19 and 14 days when the binding protein is administered to a CD-1 mouse subject at 4-5 mg/kg.
 13. The binding protein of claim 1, wherein the binding protein is cleared from a Sprague-Dawley rat subject at 0.21 and 0.24 mL/h/kg when administered at 4-5 mg/kg.
 14. The binding protein of claim 1, wherein the volume of distribution of the binding protein is between 78 and 71 mL/kg when the binding protein is administered to a Sprague-Dawley rat subject at 4-5 mg/kg.
 15. The binding protein of claim 1, wherein the half-life of the binding protein is between 19 and 12 days when the binding protein is administered to a Sprague-Dawley rat subject at 4-5 mg/kg. 16-26. (canceled)
 27. A binding protein that specifically binds to both IL-1α and IL-1β, comprising first and second polypeptide chains, wherein said first polypeptide chain comprises a first VD1-(X1)n-VD2-C-(X2)n, wherein: VD 1 is a first heavy chain variable domain; VD2 is a second heavy chain variable domain; C is a heavy chain constant domain; X1 is a linker with the proviso that it is not CH1; X2 is an Fc region; and n is independently 0 or 1; wherein said second polypeptide chain comprises a second VD1-(X1)n-VD2-C-(X2)n, wherein: VD1 is a first light chain variable domain; VD2 is a second light chain variable domain; C is a light chain constant domain; X1 is a linker with the proviso that it is not CH1; X2 does not comprise an Fc region; and n is independently 0 or 1; wherein, in said first polypeptide chain, VD1 comprises an amino acid sequence 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 31, 32, 33 and 34; and VD2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 128; wherein, in said second polypeptide chain, VD1 comprises an amino acid sequence 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 37, 38, 39 and 40; and VD2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:129; wherein the VD1 of the first polypeptide chain and the VD1 of the second polypeptide chain form a IL-1β antigen binding domain; and wherein the VD2 of the first polypeptide chain and the VD2 of the second polypeptide chain form a IL-1α antigen binding domain.
 28. The binding protein according to claim 27, wherein: said first polypeptide chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 46, 48, 50, 52, and
 54. 29. The binding protein according to claim 27, wherein: said second polypeptide chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 47, 49, 51, 53, and
 55. 30. The binding protein according to claim 27, wherein in said first polypeptide chain, VD1 comprises a VH amino acid sequence found in Table 3b (SEQ ID NOs 152-292) and in said second polypeptide chain, VD1 comprises a VL amino acid sequence found in Table 3b (SEQ ID NOs 293-419); wherein the VD1 of the first polypeptide chain and the VD1 of the second polypeptide chain form a IL-1β antigen binding domain; and wherein the VD2 of the first polypeptide chain and the VD2 of the second polypeptide chain form a IL-1α antigen binding domain.
 31. The binding protein according to claim 27, wherein X1 or X2 is an amino acid sequence selected from the group consisting of SEQ ID NOs:42 and
 43. 32-33. (canceled)
 34. The binding protein according to claim 27, wherein the Fc region is selected from the group consisting of an Fc region from an IgG1, an IgG1 LL to AA 234, 235 mutant, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD. 35-39. (canceled)
 40. A binding protein conjugate comprising a binding protein as described in claim 1 said binding protein conjugate further comprising an agent selected from the group consisting of: an immunoadhesion molecule, an imaging agent, a therapeutic agent, and a cytotoxic agent. 41-50. (canceled)
 51. A pharmaceutical composition comprising a binding protein as described in claim 1 and a pharmaceutically acceptable carrier. 52-57. (canceled)
 58. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain amino acid sequence of a binding protein described in claim
 1. 59. A vector comprising one or more isolated nucleic acid molecules according to claim
 58. 60-70. (canceled)
 71. A method of producing a binding protein, comprising culturing a host cell comprising the vector described in claim 59 in culture medium under conditions sufficient to produce the binding protein. 72-74. (canceled)
 75. A method for treating a subject for a disorder in which IL-1 activity is detrimental comprising administering to the subject a binding protein as described in claim 1 or a crystal thereof such that treatment is achieved.
 76. (canceled)
 77. A binding protein conjugate comprising a binding protein as described in claim 27, said binding protein conjugate further comprising an agent selected from the group consisting of an immunoadhesion molecule, an imaging agent, a therapeutic agent, and a cytotoxic agent.
 78. A pharmaceutical composition comprising a binding protein as described in claim 27, and a pharmaceutically acceptable carrier.
 79. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain amino acid sequence of a binding protein described in claim
 27. 80. A vector comprising one or more isolated nucleic acid molecules according to claim
 79. 81. A method of producing a binding protein, comprising the step of culturing a host cell comprising the vector described in claim 80 in culture medium under conditions sufficient to produce the binding protein.
 82. A method for treating a subject for a disorder in which IL-1 activity is detrimental comprising administering to the subject a binding protein as described in claim 27, such that treatment is achieved. 